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1
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Bergman S, Birk C, Holmqvist E. ProQ prevents mRNA degradation through inhibition of poly(A) polymerase. Nucleic Acids Res 2025; 53:gkaf103. [PMID: 40036335 DOI: 10.1093/nar/gkaf103] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2024] [Revised: 01/29/2025] [Accepted: 02/09/2025] [Indexed: 03/06/2025] Open
Abstract
The RNA-binding protein ProQ interacts with many transcripts in the bacterial cell. ProQ binding is associated with increased messenger RNA (mRNA) levels, but a mechanistic explanation for this effect has been lacking. In Salmonella Typhimurium, ProQ affects key traits associated with infection, including motility and intracellular survival. However, the direct links between ProQ activity and these phenotypes are not well understood. Here, we demonstrate that ProQ promotes biofilm formation, another virulence-associated phenotype. This effect is strictly dependent on sigma factor RpoS. ProQ increases both RpoS protein and rpoS mRNA levels, but neither affects rpoS transcription nor translation. The rpoS mRNA is a ProQ target, and expression of the rpoS 3'UTR alone is strongly dependent on ProQ. RpoS expression becomes independent of ProQ in strains lacking poly(A) polymerase I (PAPI), indicating that ProQ protects against 3' end-dependent decay. Indeed, purified ProQ inhibits PAPI-mediated polyadenylation at RNA 3' ends. Finally, PAPI is required for ProQ's effect on expression of genes involved in biofilm, motility, osmotic stress, and virulence, indicating that inhibition of polyadenylation is a general function of ProQ.
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Affiliation(s)
- Sofia Bergman
- Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, 75124 Uppsala, Sweden
| | | | - Erik Holmqvist
- Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, 75124 Uppsala, Sweden
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2
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Bhanupriya C, Kar S. RNAi-mediated downregulation of endogenous 4-coumarate: CoA ligase activity in Sorghum bicolor to alter the lignin content, which augmented the carbohydrate content and growth. PLANTA 2025; 261:30. [PMID: 39794647 DOI: 10.1007/s00425-024-04603-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/08/2024] [Accepted: 12/27/2024] [Indexed: 01/13/2025]
Abstract
MAIN CONCLUSION This study seeks to improve the biomass extractability of Sorghum bicolor by targeting a critical enzyme, 4CL, through metabolic engineering of the lignin biosynthetic pathway at the post-transcriptional level. Sorghum bicolor L., a significant forage crop, offers a potential source of carbohydrate components for biofuel production. The high lignin content in sorghum stems often impedes the extractability of desired carbohydrate components for industrial use. Thus, the present study aimed to develop an improved variety of S. bicolor with reduced lignin through RNA interference of the endogenous 4-coumarate:CoA ligase (4CL) gene involved in the lignin biosynthetic pathway. The S. bicolor gene was isolated, characterized, and used to construct the RNAi-inducing hpRNA gene-silencing construct. Two independent transgenic sorghum lines were produced by introducing an hpRNA-induced gene-silencing cassette of the Sb4CL through Agrobacterium-mediated transformation in the shoot tips of S. bicolor. This was confirmed by PCR amplification of the hygromycin-resistance gene and Southern hybridization. The Sb4CL gene transcript and its enzymatic activity were found to reduce to varying degrees, as shown by northern hybridization and enzyme activity in the independent transgenic samples. Endogenous Sb4CL downregulation in sorghum stem tissue correlates with reduced lignin content to a maximum range of 25%. The transfer of the transgene in the second generation was also analyzed. Decreased lignin content in the transgenic lines was compensated by increased total cell wall carbohydrates such as cellulose (36.56%) and soluble sugars (59.72%) compared to untransformed plants. The study suggests that suppressing the Sb4CL gene effectively develops better sorghum varieties with lower lignin content. This can be useful for industrial purposes, as the enhanced carbohydrate content and favorable alteration of lignin content can lead to economic benefits.
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Affiliation(s)
- Ch Bhanupriya
- Advanced Laboratory for Plant Genetic Engineering, Advanced Technology Development Centre, Indian Institute of Technology, Kharagpur, India.
| | - Satarupa Kar
- Advanced Laboratory for Plant Genetic Engineering, Advanced Technology Development Centre, Indian Institute of Technology, Kharagpur, India
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3
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Moorwood K, Smith FM, Garfield AS, Cowley M, Holt LJ, Daly RJ, Ward A. Grb7, Grb10 and Grb14, encoding the growth factor receptor-bound 7 family of signalling adaptor proteins have overlapping functions in the regulation of fetal growth and post-natal glucose metabolism. BMC Biol 2024; 22:221. [PMID: 39343875 PMCID: PMC11441139 DOI: 10.1186/s12915-024-02018-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2024] [Accepted: 09/23/2024] [Indexed: 10/01/2024] Open
Abstract
BACKGROUND The growth factor receptor bound protein 7 (Grb7) family of signalling adaptor proteins comprises Grb7, Grb10 and Grb14. Each can interact with the insulin receptor and other receptor tyrosine kinases, where Grb10 and Grb14 inhibit insulin receptor activity. In cell culture studies they mediate functions including cell survival, proliferation, and migration. Mouse knockout (KO) studies have revealed physiological roles for Grb10 and Grb14 in glucose-regulated energy homeostasis. Both Grb10 KO and Grb14 KO mice exhibit increased insulin signalling in peripheral tissues, with increased glucose and insulin sensitivity and a modestly increased ability to clear a glucose load. In addition, Grb10 strongly inhibits fetal growth such that at birth Grb10 KO mice are 30% larger by weight than wild type littermates. RESULTS Here, we generate a Grb7 KO mouse model. We show that during fetal development the expression patterns of Grb7 and Grb14 each overlap with that of Grb10. Despite this, Grb7 and Grb14 did not have a major role in influencing fetal growth, either alone or in combination with Grb10. At birth, in most respects both Grb7 KO and Grb14 KO single mutants were indistinguishable from wild type, while Grb7:Grb10 double knockout (DKO) were near identical to Grb10 KO single mutants and Grb10:Grb14 DKO mutants were slightly smaller than Grb10 KO single mutants. In the developing kidney Grb7 had a subtle positive influence on growth. An initial characterisation of Grb7 KO adult mice revealed sexually dimorphic effects on energy homeostasis, with females having a significantly smaller renal white adipose tissue depot and an enhanced ability to clear glucose from the circulation, compared to wild type littermates. Males had elevated fasted glucose levels with a trend towards smaller white adipose depots, without improved glucose clearance. CONCLUSIONS Grb7 and Grb14 do not have significant roles as inhibitors of fetal growth, unlike Grb10, and instead Grb7 may promote growth of the developing kidney. In adulthood, Grb7 contributes subtly to glucose mediated energy homeostasis, raising the possibility of redundancy between all three adaptors in physiological regulation of insulin signalling and glucose handling.
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Affiliation(s)
- Kim Moorwood
- Department of Life Sciences, University of Bath, Claverton Down, Bath, BA2 7AY, UK
| | - Florentia M Smith
- Department of Life Sciences, University of Bath, Claverton Down, Bath, BA2 7AY, UK
| | - Alastair S Garfield
- Department of Life Sciences, University of Bath, Claverton Down, Bath, BA2 7AY, UK
| | - Michael Cowley
- Department of Life Sciences, University of Bath, Claverton Down, Bath, BA2 7AY, UK
- Present Address: Department of Biological Sciences, Center for Human Health and the Environment, North Carolina State University, Campus, Box 7633, Raleigh, NC, 27695, USA
| | - Lowenna J Holt
- Cancer Research Program, Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, NSW, 2010, Australia
| | - Roger J Daly
- Cancer Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia
| | - Andrew Ward
- Department of Life Sciences, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
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4
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Yu Q, Tungsuchat-Huang T, Ioannou A, Barkan A, Maliga P. Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2024; 119:2437-2449. [PMID: 39031552 DOI: 10.1111/tpj.16930] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/06/2024] [Revised: 06/02/2024] [Accepted: 07/01/2024] [Indexed: 07/22/2024]
Abstract
Achieving optimally balanced gene expression within synthetic operons requires regulatory elements capable of providing a spectrum of expression levels. In this study, we investigate the expression of gfp reporter gene in tobacco chloroplasts, guided by variants of the plastid atpH 5' UTR, which harbors a binding site for PPR10, a protein that activates atpH at the posttranscriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maize atpH 5' UTRs in different design contexts. Notably, high GFP expression was not coupled to the stabilization of monocistronic gfp transcripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishes gfp mRNA (and GFP protein), presumably by promoting such rapid RNA cleavage and 5' exonucleolytic degradation that PPR10 had insufficient time to bind and protect gfp RNA, resulting in a substantial reduction in GFP accumulation. When combined with a mutant atpH 5' UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning of reporter gene expression across a wide range, spanning from a mere 0.02-25% of the total soluble cellular protein. These findings highlight the potential of employing cis-elements from heterologous species and expand the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.
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Affiliation(s)
- Qiguo Yu
- Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey, 08854, USA
| | | | - Alexander Ioannou
- Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey, 08854, USA
| | - Alice Barkan
- Institute of Molecular Biology, University of Oregon, Eugene, Oregon, 97403, USA
| | - Pal Maliga
- Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey, 08854, USA
- Department of Plant Biology, Rutgers University, New Brunswick, New Jersey, 08901, USA
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5
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Routhier E, Joubert A, Westbrook A, Pierre E, Lancrey A, Cariou M, Boulé JB, Mozziconacci J. In silico design of DNA sequences for in vivo nucleosome positioning. Nucleic Acids Res 2024; 52:6802-6810. [PMID: 38828788 PMCID: PMC11229325 DOI: 10.1093/nar/gkae468] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2023] [Revised: 04/24/2024] [Accepted: 05/29/2024] [Indexed: 06/05/2024] Open
Abstract
The computational design of synthetic DNA sequences with designer in vivo properties is gaining traction in the field of synthetic genomics. We propose here a computational method which combines a kinetic Monte Carlo framework with a deep mutational screening based on deep learning predictions. We apply our method to build regular nucleosome arrays with tailored nucleosomal repeat lengths (NRL) in yeast. Our design was validated in vivo by successfully engineering and integrating thousands of kilobases long tandem arrays of computationally optimized sequences which could accommodate NRLs much larger than the yeast natural NRL (namely 197 and 237 bp, compared to the natural NRL of ∼165 bp). RNA-seq results show that transcription of the arrays can occur but is not driven by the NRL. The computational method proposed here delineates the key sequence rules for nucleosome positioning in yeast and should be easily applicable to other sequence properties and other genomes.
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Affiliation(s)
- Etienne Routhier
- Laboratoire de Physique Théorique, CNRS, Sorbonne Université, Paris, France de la Matière Condensée, CNRS, Sorbonne Université, Paris, France
| | - Alexandra Joubert
- Structure et Instabilité des Génomes, Museum National d’Histoire Naturelle, CNRS, INSERM, Paris, France
| | - Alex Westbrook
- Structure et Instabilité des Génomes, Museum National d’Histoire Naturelle, CNRS, INSERM, Paris, France
| | - Edgard Pierre
- Laboratoire de Physique Théorique, CNRS, Sorbonne Université, Paris, France de la Matière Condensée, CNRS, Sorbonne Université, Paris, France
| | - Astrid Lancrey
- Structure et Instabilité des Génomes, Museum National d’Histoire Naturelle, CNRS, INSERM, Paris, France
| | - Marie Cariou
- Acquisition et Analyse de données pour l’histoire naturelle, Museum National d’Histoire Naturelle, CNRS, Paris, France
| | - Jean-Baptiste Boulé
- Structure et Instabilité des Génomes, Museum National d’Histoire Naturelle, CNRS, INSERM, Paris, France
| | - Julien Mozziconacci
- Laboratoire de Physique Théorique, CNRS, Sorbonne Université, Paris, France de la Matière Condensée, CNRS, Sorbonne Université, Paris, France
- Structure et Instabilité des Génomes, Museum National d’Histoire Naturelle, CNRS, INSERM, Paris, France
- Acquisition et Analyse de données pour l’histoire naturelle, Museum National d’Histoire Naturelle, CNRS, Paris, France
- Institut Universitaire de France, Paris, France
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6
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Kretz J, Börner J, Friedrich T, McIntosh M, Procida-Kowalski T, Gerken F, Wilhelm J, Klug G. Function of the RNA-targeting class 2 type VI CRISPR Cas system of Rhodobacter capsulatus. Front Microbiol 2024; 15:1384543. [PMID: 38741736 PMCID: PMC11089165 DOI: 10.3389/fmicb.2024.1384543] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2024] [Accepted: 04/15/2024] [Indexed: 05/16/2024] Open
Abstract
Bacteria use CRISPR Cas systems to defend against invading foreign nucleic acids, e.g., phage genomes, plasmids or mobile genetic elements. Some CRISPR Cas systems were reported to have physiological importance under a variety of abiotic stress conditions. We used physiological tests under different stress conditions and RNA-seq analyses to address the possible function of the RNA-targeting class 2 type VI CRISPR Cas system of the facultative phototrophic α-proteobacterium Rhodobacter capsulatus. Expression of the system was low under exponential non-stress conditions and high during oxidative stress, membrane stress and in stationary phase. Induction of the CRISPR Cas system in presence of a target protospacer RNA resulted in a growth arrest of R. capsulatus. RNA-seq revealed a strong alteration of the R. capsulatus transcriptome when cas13a was induced in presence of a target protospacer. RNA 5' end mapping indicated that the CRISPR Cas-dependent transcriptome remodeling is accompanied by fragmentation of cellular RNAs, e.g., for mRNAs originating from a genomic locus which encodes multiple ribosomal proteins and the RNA polymerase subunits RpoA, RpoB and RpoC. The data suggest a function of this CRISPR Cas system in regulated growth arrest, which may prevent the spread of phages within the population.
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Affiliation(s)
- Jonas Kretz
- Institute of Microbiology and Molecular Biology, Justus-Liebig-University, Giessen, Germany
| | - Janek Börner
- Institute of Microbiology and Molecular Biology, Justus-Liebig-University, Giessen, Germany
| | - Tobias Friedrich
- Institute of Biochemistry, Justus-Liebig-University, Giessen, Germany
- Biomedical Informatics and Systems Medicine, Justus-Liebig-University, Giessen, Germany
| | - Matthew McIntosh
- Institute of Microbiology and Molecular Biology, Justus-Liebig-University, Giessen, Germany
| | | | - Florian Gerken
- Institute of Microbiology and Molecular Biology, Justus-Liebig-University, Giessen, Germany
| | - Jochen Wilhelm
- Institute for Lung Health, Justus-Liebig-University, Giessen, Germany
| | - Gabriele Klug
- Institute of Microbiology and Molecular Biology, Justus-Liebig-University, Giessen, Germany
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7
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Cormier N, Worsham AE, Rich KA, Hardy DM. SMA20/PMIS2 Is a Rapidly Evolving Sperm Membrane Alloantigen with Possible Species-Divergent Function in Fertilization. Int J Mol Sci 2024; 25:3652. [PMID: 38612464 PMCID: PMC11011635 DOI: 10.3390/ijms25073652] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2024] [Revised: 03/05/2024] [Accepted: 03/19/2024] [Indexed: 04/14/2024] Open
Abstract
Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a-Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals.
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Affiliation(s)
- Nathaly Cormier
- Department of Biological Sciences, University of Wisconsin-Whitewater, Whitewater, WI 53190, USA
| | - Asha E. Worsham
- Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; (A.E.W.); (K.A.R.)
| | - Kinsey A. Rich
- Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; (A.E.W.); (K.A.R.)
| | - Daniel M. Hardy
- Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; (A.E.W.); (K.A.R.)
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8
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Ahmad W, Baby J, Gull B, Mustafa F. Liquid and Solid Hybridization Methods to Detect RNAs. Methods Mol Biol 2024; 2822:125-141. [PMID: 38907916 DOI: 10.1007/978-1-0716-3918-4_10] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/24/2024]
Abstract
Northern blotting (NB) has been a long-standing method for RNA detection. However, its labor-intensive nature, reliance on high-quality RNA, and use of radioactivity have diminished its appeal over time. Nevertheless, the emergence of microRNAs (miRNAs) has reignited the demand for sensitive and quantitative NB techniques. We have recently developed cost-effective and rapid protocols for RNA detection using solid and liquid hybridization (LH) techniques which exhibit high sensitivity without the need for radioactive or specialized reagents like locked nucleic acid (LNA) probes. Our assays incorporate biotinylated probes and improved techniques for probe hybridization, transfer, cross-linking, and signal enhancement. We demonstrate that while NB is sensitive in detecting mRNAs and small RNAs, our LH protocol efficiently detects these as well as miRNAs at lower amounts of RNA, achieving higher sensitivity comparable to radiolabeled probes. Compared to NB, LH offers benefits of speed, sensitivity, and specificity in detecting mRNAs, small RNAs, and miRNAs.
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Affiliation(s)
- Waqar Ahmad
- Department of Biochemistry & Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates (UAE) University, Al Ain, United Arab Emirates
| | - Jasmin Baby
- Department of Biochemistry & Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates (UAE) University, Al Ain, United Arab Emirates
- Department of Microbiology & Immunology, The University of British Columbia, Vancouver, Canada
| | - Bushra Gull
- Department of Biochemistry & Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates (UAE) University, Al Ain, United Arab Emirates
| | - Farah Mustafa
- Department of Biochemistry & Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates (UAE) University, Al Ain, United Arab Emirates.
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9
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Börner J, Friedrich T, Klug G. RNase III participates in control of quorum sensing, pigmentation and oxidative stress resistance in Rhodobacter sphaeroides. Mol Microbiol 2023; 120:874-892. [PMID: 37823424 DOI: 10.1111/mmi.15181] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2023] [Revised: 09/21/2023] [Accepted: 09/26/2023] [Indexed: 10/13/2023]
Abstract
RNase III is a dsRNA-specific endoribonuclease, highly conserved in bacteria and eukarya. In this study, we analysed the effects of inactivation of RNase III on the transcriptome and the phenotype of the facultative phototrophic α-proteobacterium Rhodobacter sphaeroides. RNA-seq revealed an unexpectedly high amount of genes with increased expression located directly downstream to the rRNA operons. Chromosomal insertion of additional transcription terminators restored wild type-like expression of the downstream genes, indicating that RNase III may modulate the rRNA transcription termination in R. sphaeroides. Furthermore, we identified RNase III as a major regulator of quorum-sensing autoinducer synthesis in R. sphaeroides. It negatively controls the expression of the autoinducer synthase CerI by reducing cerI mRNA stability. In addition, RNase III inactivation caused altered resistance against oxidative stress and impaired formation of photosynthetically active pigment-protein complexes. We also observed an increase in the CcsR small RNAs that were previously shown to promote resistance to oxidative stress. Taken together, our data present interesting insights into RNase III-mediated regulation and expand the knowledge on the function of this important enzyme in bacteria.
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Affiliation(s)
- Janek Börner
- Institute of Microbiology and Molecular Biology, Justus-Liebig-University Giessen, Giessen, Germany
| | - Tobias Friedrich
- Biomedical Informatics and Systems Medicine, Justus-Liebig-University Giessen, Giessen, Germany
- Institute of Biochemistry, Justus-Liebig-University Giessen, Giessen, Germany
| | - Gabriele Klug
- Institute of Microbiology and Molecular Biology, Justus-Liebig-University Giessen, Giessen, Germany
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10
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Ortega JA, Sasselli IR, Boccitto M, Fleming AC, Fortuna TR, Li Y, Sato K, Clemons TD, Mckenna ED, Nguyen TP, Anderson EN, Asin J, Ichida JK, Pandey UB, Wolin SL, Stupp SI, Kiskinis E. CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology. SCIENCE ADVANCES 2023; 9:eadf7997. [PMID: 37948524 PMCID: PMC10637751 DOI: 10.1126/sciadv.adf7997] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/13/2022] [Accepted: 10/11/2023] [Indexed: 11/12/2023]
Abstract
Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA "bait" oligonucleotides restore poly-GR-associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.
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Affiliation(s)
- Juan A. Ortega
- The Ken & Ruth Davee Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
- Department of Pathology and Experimental Therapy, Institute of Neurosciences, University of Barcelona, Barcelona 08907, Spain
| | - Ivan R. Sasselli
- Department of Chemistry, Northwestern University, Evanston, IL 60208, USA
- Simpson Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL 60611, USA
- Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), Donostia-San Sebastián 20014, Spain
- Centro de Fisica de Materiales (CFM), CSIC-UPV/EHU, 20018 San Sebastián, Spain
| | - Marco Boccitto
- RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA
| | - Andrew C. Fleming
- The Ken & Ruth Davee Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
| | - Tyler R. Fortuna
- Department of Pediatrics, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA
| | - Yichen Li
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Kohei Sato
- Department of Chemistry, Northwestern University, Evanston, IL 60208, USA
- Simpson Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL 60611, USA
| | - Tristan D. Clemons
- Department of Chemistry, Northwestern University, Evanston, IL 60208, USA
- Simpson Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL 60611, USA
| | - Elizabeth D. Mckenna
- The Ken & Ruth Davee Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
| | - Thao P. Nguyen
- The Ken & Ruth Davee Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
| | - Eric N. Anderson
- Department of Pediatrics, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA
| | - Jesus Asin
- Department of Statistical Methods, School of Engineering, University of Zaragoza, Zaragoza 50018, Spain
| | - Justin K. Ichida
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Udai B. Pandey
- Department of Pediatrics, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA
| | - Sandra L. Wolin
- RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA
| | - Samuel I. Stupp
- Department of Chemistry, Northwestern University, Evanston, IL 60208, USA
- Simpson Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL 60611, USA
- Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA
- Department of Materials Science and Engineering, Northwestern University, Evanston, IL 60208, USA
- Department of Medicine, Northwestern University, Chicago, IL 60611, USA
| | - Evangelos Kiskinis
- The Ken & Ruth Davee Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
- Simpson Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL 60611, USA
- Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
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11
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Sauer M, Zhao J, Park M, Khangura RS, Dilkes BP, Poethig RS. Identification of the Teopod1, Teopod2, and Early Phase Change genes in maize. G3 (BETHESDA, MD.) 2023; 13:jkad179. [PMID: 37548268 PMCID: PMC10542106 DOI: 10.1093/g3journal/jkad179] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/26/2023] [Revised: 05/26/2023] [Accepted: 08/01/2023] [Indexed: 08/08/2023]
Abstract
Teopod1 (Tp1), Teopod2 (Tp2), and Early phase change (Epc) have profound effects on the timing of vegetative phase change in maize. Gain-of-function mutations in Tp1 and Tp2 delay all known phase-specific vegetative traits, whereas loss-of-function mutations in Epc accelerate vegetative phase change and cause shoot abortion in some genetic backgrounds. Here, we show that Tp1 and Tp2 likely represent cis-acting mutations that cause the overexpression of Zma-miR156j and Zma-miR156h, respectively. Epc is the maize ortholog of HASTY, an Arabidopsis gene that stabilizes miRNAs and promotes their intercellular movement. Consistent with its pleiotropic phenotype and epistatic interaction with Tp1 and Tp2, epc reduces the levels of miR156 and several other miRNAs.
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Affiliation(s)
- Matt Sauer
- Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Jianfei Zhao
- Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Meeyeon Park
- Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Rajdeep S Khangura
- Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA
| | - Brian P Dilkes
- Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA
| | - R Scott Poethig
- Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
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12
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Watanabe K, Fujita M, Okamoto K, Yoshioka H, Moriwaki M, Tagashira H, Awazu A, Yamamoto T, Sakamoto N. The crucial role of CTCF in mitotic progression during early development of sea urchin. Dev Growth Differ 2023; 65:395-407. [PMID: 37421304 DOI: 10.1111/dgd.12875] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2023] [Revised: 07/03/2023] [Accepted: 07/04/2023] [Indexed: 07/10/2023]
Abstract
CCCTC-binding factor (CTCF), an insulator protein with 11 zinc fingers, is enriched at the boundaries of topologically associated domains (TADs) in eukaryotic genomes. In this study, we isolated and analyzed the cDNAs encoding HpCTCF, the CTCF homolog in the sea urchin Hemicentrotus pulcherrimus, to investigate its expression patterns and functions during the early development of sea urchin. HpCTCF contains nine zinc fingers corresponding to fingers 2-10 of the vertebrate CTCF. Expression pattern analysis revealed that HpCTCF mRNA was detected at all developmental stages and in the entire embryo. Upon expressing the HpCTCF-GFP fusion protein in early embryos, we observed its uniform distribution within interphase nuclei. However, during mitosis, it disappeared from the chromosomes and subsequently reassembled on the chromosome during telophase. Moreover, the morpholino-mediated knockdown of HpCTCF resulted in mitotic arrest during the morula to blastula stage. Most of the arrested chromosomes were not phospholylated at serine 10 of histone H3, indicating that mitosis was arrested at the telophase by HpCTCF depletion. Furthermore, impaired sister chromatid segregation was observed using time-lapse imaging of HpCTCF-knockdown embryos. Thus, HpCTCF is essential for mitotic progression during the early development of sea urchins, especially during the telophase-to-interphase transition. However, the normal development of pluteus larvae in CRISPR-mediated HpCTCF-knockout embryos suggests that disruption of zygotic HpCTCF expression has little effect on embryonic and larval development.
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Affiliation(s)
- Kaichi Watanabe
- Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Japan
| | - Megumi Fujita
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan
| | - Kazuko Okamoto
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan
- Amphibian Research Center, Hiroshima University, Higashi-Hiroshima, Japan
| | - Hajime Yoshioka
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan
| | - Miki Moriwaki
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan
| | - Hideki Tagashira
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan
| | - Akinori Awazu
- Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Japan
- Research Center for the Mathematics on Chromatin Live Dynamics, Hiroshima University, Higashi-Hiroshima, Japan
| | - Takashi Yamamoto
- Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Japan
- Research Center for the Mathematics on Chromatin Live Dynamics, Hiroshima University, Higashi-Hiroshima, Japan
| | - Naoaki Sakamoto
- Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Japan
- Research Center for the Mathematics on Chromatin Live Dynamics, Hiroshima University, Higashi-Hiroshima, Japan
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13
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Ragunathan PT, Ng Kwan Lim E, Ma X, Massé E, Vanderpool CK. Mechanisms of Regulation of Cryptic Prophage-Encoded Gene Products in Escherichia coli. J Bacteriol 2023; 205:e0012923. [PMID: 37439671 PMCID: PMC10448788 DOI: 10.1128/jb.00129-23] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2023] [Accepted: 06/12/2023] [Indexed: 07/14/2023] Open
Abstract
The dicBF operon of Qin cryptic prophage in Escherichia coli K-12 encodes the small RNA (sRNA) DicF and small protein DicB, which regulate host cell division and are toxic when overexpressed. While new functions of DicB and DicF have been identified in recent years, the mechanisms controlling the expression of the dicBF operon have remained unclear. Transcription from dicBp, the major promoter of the dicBF operon, is repressed by DicA. In this study, we discovered that transcription of the dicBF operon and processing of the polycistronic mRNA is regulated by multiple mechanisms. DicF sRNA accumulates during stationary phase and is processed from the polycistronic dicBF mRNA by the action of both RNase III and RNase E. DicA-mediated transcriptional repression of dicBp can be relieved by an antirepressor protein, Rem, encoded on the Qin prophage. Ectopic production of Rem results in cell filamentation due to strong induction of the dicBF operon, and filamentation is mediated by DicF and DicB. Spontaneous derepression of dicBp occurs in a subpopulation of cells independent of the antirepressor. This phenomenon is reminiscent of the bistable switch of λ phage with DicA and DicC performing functions similar to those of CI and Cro, respectively. Additional experiments demonstrate stress-dependent induction of the dicBF operon. Collectively, our results illustrate that toxic genes carried on cryptic prophages are subject to layered mechanisms of control, some that are derived from the ancestral phage and some that are likely later adaptations. IMPORTANCE Cryptic or defective prophages have lost genes necessary to excise from the bacterial chromosome and produce phage progeny. In recent years, studies have found that cryptic prophage gene products influence diverse aspects of bacterial host cell physiology. However, to obtain a complete understanding of the relationship between cryptic prophages and the host bacterium, identification of the environmental, host, or prophage-encoded factors that induce the expression of cryptic prophage genes is crucial. In this study, we examined the regulation of a cryptic prophage operon in Escherichia coli encoding a small RNA and a small protein that are involved in inhibiting bacterial cell division, altering host metabolism, and protecting the host bacterium from phage infections.
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Affiliation(s)
- Preethi T. Ragunathan
- Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
| | - Evelyne Ng Kwan Lim
- Department of Biochemistry and Functional Genomics, University of Sherbrooke, Sherbrooke, Québec, Canada
| | - Xiangqian Ma
- Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
| | - Eric Massé
- Department of Biochemistry and Functional Genomics, University of Sherbrooke, Sherbrooke, Québec, Canada
| | - Carin K. Vanderpool
- Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
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14
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Grützner J, Börner J, Jäger A, Klug G. The Small RNA-Binding Protein CcaF1 Promotes Formation of Photosynthetic Complexes in Rhodobacter sphaeroides. Int J Mol Sci 2023; 24:ijms24119515. [PMID: 37298460 DOI: 10.3390/ijms24119515] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2023] [Revised: 05/24/2023] [Accepted: 05/25/2023] [Indexed: 06/12/2023] Open
Abstract
In natural habitats, bacteria frequently need to adapt to changing environmental conditions. Regulation of transcription plays an important role in this process. However, riboregulation also contributes substantially to adaptation. Riboregulation often acts at the level of mRNA stability, which is determined by sRNAs, RNases, and RNA-binding proteins. We previously identified the small RNA-binding protein CcaF1, which is involved in sRNA maturation and RNA turnover in Rhodobacter sphaeroides. Rhodobacter is a facultative phototroph that can perform aerobic and anaerobic respiration, fermentation, and anoxygenic photosynthesis. Oxygen concentration and light conditions decide the pathway for ATP production. Here, we show that CcaF1 promotes the formation of photosynthetic complexes by increasing levels of mRNAs for pigment synthesis and for some pigment-binding proteins. Levels of mRNAs for transcriptional regulators of photosynthesis genes are not affected by CcaF1. RIP-Seq analysis compares the binding of CcaF1 to RNAs during microaerobic and photosynthetic growth. The stability of the pufBA mRNA for proteins of the light-harvesting I complex is increased by CcaF1 during phototrophic growth but decreased during microaerobic growth. This research underlines the importance of RNA-binding proteins in adaptation to different environments and demonstrates that an RNA-binding protein can differentially affect its binding partners in dependence upon growth conditions.
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Affiliation(s)
- Julian Grützner
- Institute of Microbiology and Molecular Biology, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
| | - Janek Börner
- Institute of Microbiology and Molecular Biology, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
| | - Andreas Jäger
- Institute of Microbiology and Molecular Biology, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
| | - Gabriele Klug
- Institute of Microbiology and Molecular Biology, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
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15
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Stenum TS, Kumar AD, Sandbaumhüter FA, Kjellin J, Jerlström-Hultqvist J, Andrén PE, Koskiniemi S, Jansson E, Holmqvist E. RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins. Nucleic Acids Res 2023; 51:4572-4587. [PMID: 36987847 PMCID: PMC10201417 DOI: 10.1093/nar/gkad216] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2022] [Revised: 03/03/2023] [Accepted: 03/16/2023] [Indexed: 03/30/2023] Open
Abstract
RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.
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Affiliation(s)
- Thomas Søndergaard Stenum
- Microbiology and Immunology, Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Box 596, 75124 Uppsala, Sweden
| | - Ankith D Kumar
- Microbiology and Immunology, Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Box 596, 75124 Uppsala, Sweden
| | - Friederike A Sandbaumhüter
- Medical Mass Spectrometry, Department of Pharmaceutical Biosciences, Biomedical Centre, Uppsala University, Box 591, 75124 Uppsala, Sweden
| | - Jonas Kjellin
- Microbiology and Immunology, Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Box 596, 75124 Uppsala, Sweden
| | - Jon Jerlström-Hultqvist
- Microbiology and Immunology, Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Box 596, 75124 Uppsala, Sweden
| | - Per E Andrén
- Medical Mass Spectrometry, Department of Pharmaceutical Biosciences, Biomedical Centre, Uppsala University, Box 591, 75124 Uppsala, Sweden
- Science for Life Laboratory, Spatial Mass Spectrometry, Biomedical Centre, Uppsala University, Box 591, 75124 Uppsala, Sweden
| | - Sanna Koskiniemi
- Microbiology and Immunology, Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Box 596, 75124 Uppsala, Sweden
| | - Erik T Jansson
- Medical Mass Spectrometry, Department of Pharmaceutical Biosciences, Biomedical Centre, Uppsala University, Box 591, 75124 Uppsala, Sweden
| | - Erik Holmqvist
- Microbiology and Immunology, Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Box 596, 75124 Uppsala, Sweden
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16
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Schwartz AM, Marcotte HA, Johnson CN. Evaluation of Alternative Colony Hybridization Methods for Pathogenic Vibrios. Foods 2023; 12:foods12071472. [PMID: 37048292 PMCID: PMC10093671 DOI: 10.3390/foods12071472] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Revised: 03/23/2023] [Accepted: 03/28/2023] [Indexed: 04/03/2023] Open
Abstract
Vibrios, such as Vibrio parahaemolyticus, are naturally occurring halophilic bacteria that are a major cause of foodborne illness. Because of their autochthonous nature, managing vibrio levels in marine and estuarine environments is impossible. Instead, it is crucial to reliably enumerate their abundance to minimize human exposure. One method of achieving this is the direct plating/colony hybridization (DP/CH) method, which has been used to efficiently quantify pathogenic vibrios in oysters and other seafood products. Although successful, the method relies on proprietary resources. We examined alternative approaches, assessed the influence of the reagent suppliers’ source on enumeration accuracy, and made experimental adjustments that maximized efficiency, sensitivity, and specificity. We report here that in-house conjugation via Cell Mosaic is a viable alternative to the previously available sole-source distributor of the alkaline phosphatase-conjugated probes used to enumerate vibrios in oysters. We also report that milk was a viable alternative as a blocking reagent, pH must be eight, an orbital shaker was a viable alternative to a water bath, and narrow polypropylene containers were a viable alternative to Whirl-Pak bags. These modifications will be crucial to scientists enumerating vibrios and other pathogens in food products.
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17
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Sanpedro-Luna JA, Jacinto-Vázquez JJ, Anastacio-Marcelino E, Posadas-Gutiérrez CM, Olmos-Pineda I, González-Bernal JA, Carcaño-Montiel M, Vega-Alvarado L, Vázquez-Cruz C, Sánchez-Alonso P. Telomerase RNA plays a major role in the completion of the life cycle in Ustilago maydis and shares conserved domains with other Ustilaginales. PLoS One 2023; 18:e0281251. [PMID: 36952474 PMCID: PMC10035886 DOI: 10.1371/journal.pone.0281251] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Accepted: 01/18/2023] [Indexed: 03/25/2023] Open
Abstract
The RNA subunit of telomerase is an essential component whose primary sequence and length are poorly conserved among eukaryotic organisms. The phytopathogen Ustilago maydis is a dimorphic fungus of the order Ustilaginales. We analyzed several species of Ustilaginales to computationally identify the TElomere RNA (TER) gene ter1. To confirm the identity of the TER gene, we disrupted the gene and characterized telomerase-negative mutants. Similar to catalytic TERT mutants, ter1Δ mutants exhibit phenotypes of growth delay, telomere shortening and low replicative potential. ter1-disrupted mutants were unable to infect maize seedlings in heterozygous crosses and showed defects such as cell cycle arrest and segregation failure. We concluded that ter1, which encodes the TER subunit of the telomerase of U. maydis, have similar and perhaps more extensive functions than trt1.
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Affiliation(s)
- Juan Antonio Sanpedro-Luna
- Instituto de Ciencias, Posgrado en Microbiología, Benemérita Universidad Autónoma de Puebla, Puebla, México
| | - José Juan Jacinto-Vázquez
- Instituto de Ciencias, Posgrado en Microbiología, Benemérita Universidad Autónoma de Puebla, Puebla, México
| | - Estela Anastacio-Marcelino
- Instituto de Ciencias, Centro de Investigaciones Microbiológicas, Benemérita Universidad Autónoma de Puebla, Puebla, México
| | | | - Iván Olmos-Pineda
- Facultad de Ciencias de la Computación, Benemérita Universidad Autónoma de Puebla, Puebla, México
| | - Jesús Antonio González-Bernal
- Department of Computer Science and Engineering, The University of Texas Arlington, Arlington, Texas, United States of America
| | - Moisés Carcaño-Montiel
- Instituto de Ciencias, Centro de Investigaciones Microbiológicas, Benemérita Universidad Autónoma de Puebla, Puebla, México
| | - Leticia Vega-Alvarado
- Instituto de Ciencias Aplicadas y Tecnología, Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de México, México, México
| | - Candelario Vázquez-Cruz
- Instituto de Ciencias, Posgrado en Microbiología, Benemérita Universidad Autónoma de Puebla, Puebla, México
- Instituto de Ciencias, Centro de Investigaciones Microbiológicas, Benemérita Universidad Autónoma de Puebla, Puebla, México
| | - Patricia Sánchez-Alonso
- Instituto de Ciencias, Posgrado en Microbiología, Benemérita Universidad Autónoma de Puebla, Puebla, México
- Instituto de Ciencias, Centro de Investigaciones Microbiológicas, Benemérita Universidad Autónoma de Puebla, Puebla, México
- * E-mail:
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18
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Guerra-Slompo E, Cesaro G, Guimarães B, Zanchin N. Dissecting Trypanosoma brucei RRP44 function in the maturation of segmented ribosomal RNA using a regulated genetic complementation system. Nucleic Acids Res 2023; 51:396-419. [PMID: 36610751 PMCID: PMC9841430 DOI: 10.1093/nar/gkac1217] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 11/29/2022] [Accepted: 01/03/2023] [Indexed: 01/09/2023] Open
Abstract
Trypanosoma brucei belongs to a group of protozoans presenting fragmented large subunit rRNA. Its LSU rRNA equivalent to the 25S/28S rRNA of other eukaryotes is split into six fragments, requiring additional processing for removal of the extra spacer sequences. We have used a genetic complementation strategy to further investigate the T. brucei RRP44 nuclease in pre-rRNA maturation. TbRRP44 contains both a PIN and a RNB domain whose homologues are found in association with the exosome complex. We found that the exonucleolytic activity of the RNB domain as well as the physical presence of the PIN domain are essential for TbRRP44 function, while a catalytic site mutation in the PIN domain has no detectable effect on cell growth. A new endonucleolytic cleavage site in ITS1 was identified. In addition to the 5.8S rRNA 3'-end maturation, TbRRP44 is required for degradation of the excised 5'-ETS and for removal of part of ITS1 during maturation of the 18S rRNA 3'-end. TbRRP44 deficiency leads to accumulation of many LSU intermediate precursors, most of them not detected in control cells. TbRRP44 is also required for U3 snoRNA and spliced leader processing, indicating that TbRRP44 may have a wide role in RNA processing in T. brucei.
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Affiliation(s)
- Eloise Pavão Guerra-Slompo
- Carlos Chagas Institute, Oswaldo Cruz Foundation, FIOCRUZ, R. Prof. Algacyr Munhoz Mader 3775, 81350-010, Curitiba-PR, Brazil
| | - Giovanna Cesaro
- Carlos Chagas Institute, Oswaldo Cruz Foundation, FIOCRUZ, R. Prof. Algacyr Munhoz Mader 3775, 81350-010, Curitiba-PR, Brazil,Biochemistry Postgraduate Program, Federal University of Paraná, Curitiba-PR, Brazil
| | - Beatriz Gomes Guimarães
- Carlos Chagas Institute, Oswaldo Cruz Foundation, FIOCRUZ, R. Prof. Algacyr Munhoz Mader 3775, 81350-010, Curitiba-PR, Brazil,Biochemistry Postgraduate Program, Federal University of Paraná, Curitiba-PR, Brazil
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19
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Carrier MC, Lalaouna D, Massé E. Hfq protein and GcvB small RNA tailoring of oppA target mRNA to levels allowing translation activation by MicF small RNA in Escherichia coli. RNA Biol 2023; 20:59-76. [PMID: 36860088 PMCID: PMC9988348 DOI: 10.1080/15476286.2023.2179582] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/03/2023] Open
Abstract
Traffic of molecules across the bacterial membrane mainly relies on porins and transporters, whose expression must adapt to environmental conditions. To ensure bacterial fitness, synthesis and assembly of functional porins and transporters are regulated through a plethora of mechanisms. Among them, small regulatory RNAs (sRNAs) are known to be powerful post-transcriptional regulators. In Escherichia coli, the MicF sRNA is known to regulate only four targets, a very narrow targetome for a sRNA responding to various stresses, such as membrane stress, osmotic shock, or thermal shock. Using an in vivo pull-down assay combined with high-throughput RNA sequencing, we sought to identify new targets of MicF to better understand its role in the maintenance of cellular homoeostasis. Here, we report the first positively regulated target of MicF, the oppA mRNA. The OppA protein is the periplasmic component of the Opp ATP-binding cassette (ABC) oligopeptide transporter and regulates the import of short peptides, some of them bactericides. Mechanistic studies suggest that oppA translation is activated by MicF through a mechanism of action involving facilitated access to a translation-enhancing region in oppA 5'UTR. Intriguingly, MicF activation of oppA translation depends on cross-regulation by negative trans-acting effectors, the GcvB sRNA and the RNA chaperone protein Hfq.
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Affiliation(s)
- Marie-Claude Carrier
- Department of Biochemistry and Functional Genomics, RNA Group, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - David Lalaouna
- Department of Biochemistry and Functional Genomics, RNA Group, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Eric Massé
- Department of Biochemistry and Functional Genomics, RNA Group, Université de Sherbrooke, Sherbrooke, Québec, Canada
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20
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The PNUTS-PP1 complex acts as an intrinsic barrier to herpesvirus KSHV gene expression and replication. Nat Commun 2022; 13:7447. [PMID: 36460671 PMCID: PMC9718767 DOI: 10.1038/s41467-022-35268-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2022] [Accepted: 11/24/2022] [Indexed: 12/03/2022] Open
Abstract
Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-PP1 complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion enhances global KSHV gene expression and overall viral replication. Mechanistically, PNUTS requires PP1 interaction, binds viral RNAs downstream of polyadenylation sites, and restricts transcription readthrough of viral genes. Surprisingly, PNUTS also represses productive elongation at the 5´ ends of the KSHV reporter and the KSHV T1.4 RNA. From these data, we conclude that PNUTS' activity constitutes an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.
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21
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Lee S, Jo SH, Hong CE, Lee J, Cha B, Park JM. Plastid methylerythritol phosphate pathway participates in the hypersensitive response-related cell death in Nicotiana benthamiana. FRONTIERS IN PLANT SCIENCE 2022; 13:1032682. [PMID: 36388595 PMCID: PMC9645581 DOI: 10.3389/fpls.2022.1032682] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Accepted: 10/07/2022] [Indexed: 06/16/2023]
Abstract
Programmed cell death (PCD), a characteristic feature of hypersensitive response (HR) in plants, is an important cellular process often associated with the defense response against pathogens. Here, the involvement of LytB, a gene encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase that participates in the final step of the plastid methylerythritol phosphate (MEP) pathway, in plant HR cell death was studied. In Nicotiana benthmiana plants, silencing of the NbLytB gene using virus-induced gene silencing (VIGS) caused plant growth retardation and albino leaves with severely malformed chloroplasts. In NbLytB-silenced plants, HR-related cell death mediated by the expression of either the human proapoptotic protein gene Bax or an R gene with its cognate Avr effector gene was inhibited, whereas that induced by the nonhost pathogen Pseudomonas syringae pv. syringae 61 was enhanced. To dissect the isoprenoid pathway and avoid the pleiotropic effects of VIGS, chemical inhibitors that specifically inhibit isoprenoid biosynthesis in plants were employed. Treatment of N. benthamiana plants with fosmidomycin, a specific inhibitor of the plastid MEP pathway, effectively inhibited HR-related PCD, whereas treatment with mevinolin (a cytoplasmic mevalonate pathway inhibitor) and fluridone (a carotenoid biosynthesis inhibitor) did not. Together, these results suggest that the MEP pathway as well as reactive oxygen species (ROS) generation in the chloroplast play an important role in HR-related PCD, which is not displaced by the cytosolic isoprenoid biosynthesis pathway.
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Affiliation(s)
- Sanghun Lee
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, South Korea
- Department of Plant Medicine, Chungbuk National University, Cheongju, South Korea
| | - Sung Hee Jo
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, South Korea
| | - Chi Eun Hong
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, South Korea
| | - Jiyoung Lee
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, South Korea
- Biological Resource Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Jeongeup, South Korea
| | - Byeongjin Cha
- Department of Plant Medicine, Chungbuk National University, Cheongju, South Korea
| | - Jeong Mee Park
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, South Korea
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22
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Comparative transcriptome profiling and molecular marker development for oil palm fruit color. Sci Rep 2022; 12:15507. [PMID: 36109663 PMCID: PMC9478095 DOI: 10.1038/s41598-022-19890-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Accepted: 09/06/2022] [Indexed: 12/02/2022] Open
Abstract
Oil palm harvesting is normally determined by fruit exocarp color. To detect expressed sequence tag (EST)-simple sequence repeat (SSR) markers in oil palm hybrid populations, de novo transcriptomic profiling of Nigeria black and Suratthani 1 (Deli × Calabar) plants was performed. More than 46 million high-quality clean reads with a mean length of 1117 bp were generated. Functional annotation and gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) revealed that the genes were involved in fruit color development and pigment synthesis. Comparison of immature/mature DEGs indicated that nigrescent fruit color was driven by the anthocyanin biosynthesis pathway (ABP); however, the carotenoid biosynthesis pathway (CBP) was involved in the color development of both fruit types. The transcripts of both unique and different genes involved in the ABP and CBP in higher plants were highlighted for further study, especially 3GT, downstream genes in the ABP, and DEARF27 in the CBP. Additionally, SSR primer motifs, namely, 9949, discovered from the DEGs upregulated in the virescent type that encode vacuolar iron transporter (VIT), could separate the nigrescence and virescence traits of Nigeria hybrids. This novel primer has potential to be used as a molecular for further selection in breeding programs especially involving the specific genetic backgrounds described in this study.
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23
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Somssich M. The Dawn of Plant Molecular Biology: How Three Key Methodologies Paved the Way. Curr Protoc 2022; 2:e417. [PMID: 35441802 DOI: 10.1002/cpz1.417] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The adoption of Arabidopsis thaliana in the 1980s as a universal plant model finally enabled researchers to adopt and take full advantage of the molecular biology tools and methods developed in the bacterial and animal fields since the early 1970s. It further brought the plant sciences up to speed with other research fields, which had been employing widely accepted model organisms for decades. In parallel with this major development, the concurrent establishment of the plant transformation methodology and the description of the cauliflower mosaic virus (CaMV) 35S promoter enabled scientists to create robust transgenic plant lines for the first time, thereby providing a valuable tool for studying gene function. The ability to create transgenic plants launched the plant biotechnology sector, with Monsanto and Plant Genetic Systems developing the first herbicide- and pest-tolerant plants, initiating a revolution in the agricultural industry. Here I review the major developments over a less than 10-year span and demonstrate how they complemented each other to trigger a revolution in plant molecular biology and launch an era of unprecedented progress for the whole plant field. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.
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Affiliation(s)
- Marc Somssich
- School of BioSciences, University of Melbourne, Parkville, Victoria, Australia
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24
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Generative Adversarial Networks for Creating Synthetic Nucleic Acid Sequences of Cat Genome. Int J Mol Sci 2022; 23:ijms23073701. [PMID: 35409058 PMCID: PMC8998662 DOI: 10.3390/ijms23073701] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Revised: 03/23/2022] [Accepted: 03/25/2022] [Indexed: 01/04/2023] Open
Abstract
Nucleic acids are the basic units of deoxyribonucleic acid (DNA) sequencing. Every organism demonstrates different DNA sequences with specific nucleotides. It reveals the genetic information carried by a particular DNA segment. Nucleic acid sequencing expresses the evolutionary changes among organisms and revolutionizes disease diagnosis in animals. This paper proposes a generative adversarial networks (GAN) model to create synthetic nucleic acid sequences of the cat genome tuned to exhibit specific desired properties. We obtained the raw sequence data from Illumina next generation sequencing. Various data preprocessing steps were performed using Cutadapt and DADA2 tools. The processed data were fed to the GAN model that was designed following the architecture of Wasserstein GAN with gradient penalty (WGAN-GP). We introduced a predictor and an evaluator in our proposed GAN model to tune the synthetic sequences to acquire certain realistic properties. The predictor was built for extracting samples with a promoter sequence, and the evaluator was built for filtering samples that scored high for motif-matching. The filtered samples were then passed to the discriminator. We evaluated our model based on multiple metrics and demonstrated outputs for latent interpolation, latent complementation, and motif-matching. Evaluation results showed our proposed GAN model achieved 93.7% correlation with the original data and produced significant outcomes as compared to existing models for sequence generation.
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25
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Galati E, Bosio MC, Novarina D, Chiara M, Bernini GM, Mozzarelli AM, García-Rubio ML, Gómez-González B, Aguilera A, Carzaniga T, Todisco M, Bellini T, Nava GM, Frigè G, Sertic S, Horner DS, Baryshnikova A, Manzari C, D'Erchia AM, Pesole G, Brown GW, Muzi-Falconi M, Lazzaro F. VID22 counteracts G-quadruplex-induced genome instability. Nucleic Acids Res 2021; 49:12785-12804. [PMID: 34871443 PMCID: PMC8682794 DOI: 10.1093/nar/gkab1156] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2021] [Revised: 10/19/2021] [Accepted: 11/08/2021] [Indexed: 12/17/2022] Open
Abstract
Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism.
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Affiliation(s)
- Elena Galati
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
| | - Maria C Bosio
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
| | - Daniele Novarina
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
| | - Matteo Chiara
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.,Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari, Consiglio Nazionale delle Ricerche, Bari, Italy
| | - Giulia M Bernini
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
| | - Alessandro M Mozzarelli
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
| | - Maria L García-Rubio
- Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla, Seville, Spain
| | - Belén Gómez-González
- Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla, Seville, Spain
| | - Andrés Aguilera
- Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla, Seville, Spain
| | - Thomas Carzaniga
- Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, via Vanvitelli 32, 20129 Milan, Italy
| | - Marco Todisco
- Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, via Vanvitelli 32, 20129 Milan, Italy
| | - Tommaso Bellini
- Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, via Vanvitelli 32, 20129 Milan, Italy
| | - Giulia M Nava
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
| | - Gianmaria Frigè
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Via Adamello 16, 20139 Milan, Italy
| | - Sarah Sertic
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
| | - David S Horner
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.,Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari, Consiglio Nazionale delle Ricerche, Bari, Italy
| | - Anastasia Baryshnikova
- Department of Molecular Genetics and Donnelly Centre, University of Toronto, Toronto, Canada
| | - Caterina Manzari
- Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari, Consiglio Nazionale delle Ricerche, Bari, Italy
| | - Anna M D'Erchia
- Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari, Consiglio Nazionale delle Ricerche, Bari, Italy.,Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università di Bari 'A. Moro', Bari, Italy
| | - Graziano Pesole
- Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari, Consiglio Nazionale delle Ricerche, Bari, Italy.,Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università di Bari 'A. Moro', Bari, Italy
| | - Grant W Brown
- Department of Biochemistry and Donnelly Centre, University of Toronto, Ontario M5S 3E1, Toronto, Canada
| | - Marco Muzi-Falconi
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
| | - Federico Lazzaro
- Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
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26
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Zobrist JD, Martin-Ortigosa S, Lee K, Azanu MK, Ji Q, Wang K. Transformation of Teosinte ( Zea mays ssp. parviglumis) via Biolistic Bombardment of Seedling-Derived Callus Tissues. FRONTIERS IN PLANT SCIENCE 2021; 12:773419. [PMID: 34956270 PMCID: PMC8696365 DOI: 10.3389/fpls.2021.773419] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/09/2021] [Accepted: 11/09/2021] [Indexed: 05/17/2023]
Abstract
Modern maize exhibits a significantly different phenotype than its wild progenitor teosinte despite many genetic similarities. Of the many subspecies of Zea mays identified as teosinte, Zea mays ssp. parviglumis is the most closely related to domesticated maize. Understanding teosinte genes and their regulations can provide great insights into the maize domestication process and facilitate breeding for future crop improvement. However, a protocol of genetic transformation, which is essential for gene functional analyses, is not available in teosinte. In this study, we report the establishment of a robust callus induction and regeneration protocol using whorl segments of seedlings germinated from mature seeds of Zea parviglumis. We also report, for the first time, the production of fertile, transgenic teosinte plants using the particle bombardment. Using herbicide resistance genes such as mutant acetolactate synthase (Als) or bialaphos resistance (bar) as selectable markers, we achieved an average transformation frequency of 4.17% (percentage of independent transgenic events in total bombarded explants that produced callus). Expression of visual marker genes of red fluorescent protein tdTomato and β-glucuronidase (gus) could be detected in bombarded callus culture and in T1 and T2 progeny plants. The protocol established in this work provides a major enabling technology for research toward the understanding of this important plant in crop domestication.
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Affiliation(s)
- Jacob D. Zobrist
- Department of Agronomy, Iowa State University, Ames, IA, United States
- Crop Bioengineering Center, Iowa State University, Ames, IA, United States
- Interdepartmental Genetics and Genomics Major, Iowa State University, Ames, IA, United States
| | | | - Keunsub Lee
- Department of Agronomy, Iowa State University, Ames, IA, United States
- Crop Bioengineering Center, Iowa State University, Ames, IA, United States
| | - Mercy K. Azanu
- Department of Agronomy, Iowa State University, Ames, IA, United States
- Crop Bioengineering Center, Iowa State University, Ames, IA, United States
- Interdepartmental Plant Biology Major, Iowa State University, Ames, IA, United States
| | - Q Ji
- Department of Agronomy, Iowa State University, Ames, IA, United States
| | - Kan Wang
- Department of Agronomy, Iowa State University, Ames, IA, United States
- Crop Bioengineering Center, Iowa State University, Ames, IA, United States
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27
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Abstract
DNA methylation is one of the most important components of epigenetics, which plays essential roles in maintaining genome stability and regulating gene expression. In recent years, DNA methylation measuring methods have been continuously optimized. Combined with next generation sequencing technologies, these approaches have enabled the detection of genome-wide cytosine methylation at single-base resolution. In this paper, we review the development of 5-methylcytosine and its oxidized derivatives measuring methods, and recent advancement of single-cell epigenome sequencing technologies, offering more referable information for the selection and optimization of DNA methylation sequencing technologies and related research.
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28
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Cytosolic and Mitochondrial Hsp90 in Cytokinesis, Mitochondrial DNA Replication, and Drug Action in Trypanosoma brucei. Antimicrob Agents Chemother 2021; 65:e0063221. [PMID: 34424040 DOI: 10.1128/aac.00632-21] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Trypanosoma brucei subspecies cause African sleeping sickness in humans, an infection that is commonly fatal if not treated, and available therapies are limited. Previous studies have shown that heat shock protein 90 (Hsp90) inhibitors have potent and vivid activity against bloodstream-form trypanosomes. Hsp90s are phylogenetically conserved and essential catalysts that function at the crux of cell biology, where they ensure the proper folding of proteins and their assembly into multicomponent complexes. To assess the specificity of Hsp90 inhibitors and further define the role of Hsp90s in African trypanosomes, we used RNA interference (RNAi) to knock down cytosolic and mitochondrial Hsp90s (HSP83 and HSP84, respectively). Loss of either protein led to cell death, but the phenotypes were distinctly different. Depletion of cytosolic HSP83 closely mimicked the consequences of chemically depleting Hsp90 activity with inhibitor 17-AAG. In these cells, cytokinesis was severely disrupted, and segregation of the kinetoplast (the massive mitochondrial DNA structure unique to this family of eukaryotic pathogens) was impaired, leading to cells with abnormal kinetoplast DNA (kDNA) structures. Quite differently, knockdown of mitochondrial HSP84 did not impair cytokinesis but halted the initiation of new kDNA synthesis, generating cells without kDNA. These findings highlight the central role of Hsp90s in chaperoning cell cycle regulators in trypanosomes, reveal their unique function in kinetoplast replication, and reinforce their specificity and value as drug targets.
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29
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Freed-Pastor WA, Lambert LJ, Ely ZA, Pattada NB, Bhutkar A, Eng G, Mercer KL, Garcia AP, Lin L, Rideout WM, Hwang WL, Schenkel JM, Jaeger AM, Bronson RT, Westcott PMK, Hether TD, Divakar P, Reeves JW, Deshpande V, Delorey T, Phillips D, Yilmaz OH, Regev A, Jacks T. The CD155/TIGIT axis promotes and maintains immune evasion in neoantigen-expressing pancreatic cancer. Cancer Cell 2021; 39:1342-1360.e14. [PMID: 34358448 PMCID: PMC8511341 DOI: 10.1016/j.ccell.2021.07.007] [Citation(s) in RCA: 150] [Impact Index Per Article: 37.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/11/2020] [Revised: 04/26/2021] [Accepted: 07/12/2021] [Indexed: 02/08/2023]
Abstract
The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.
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Affiliation(s)
- William A Freed-Pastor
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA
| | - Laurens J Lambert
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Zackery A Ely
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Nimisha B Pattada
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Arjun Bhutkar
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - George Eng
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Kim L Mercer
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Ana P Garcia
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Lin Lin
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - William M Rideout
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - William L Hwang
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Jason M Schenkel
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
| | - Alex M Jaeger
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Roderick T Bronson
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Peter M K Westcott
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | | | | | | | - Vikram Deshpande
- Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Toni Delorey
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Devan Phillips
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Omer H Yilmaz
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Aviv Regev
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Tyler Jacks
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
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30
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Takehira K, Hayashi Y, Nozawa K, Chen L, Suzuki T, Masuta Y, Kato A, Ito H. DRD1, a SWI/SNF-like chromatin remodeling protein, regulates a heat-activated transposon in Arabidopsis thaliana. Genes Genet Syst 2021; 96:151-158. [PMID: 34373369 DOI: 10.1266/ggs.21-00005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
ONSEN is a heat-activated LTR retrotransposon in Arabidopsis thaliana. Screens to identify transcriptional regulatory factors of ONSEN revealed a SWI/SNF-like chromatin remodeling protein, DRD1, which cooperates with plant-specific RNA polymerase and is involved in RNA-directed DNA methylation. ONSEN transcript level was increased in the drd1 mutant relative to wild-type under heat stress, indicating that DRD1 plays a significant role in the silencing of activated ONSEN under the stress condition. The transcript level of HsfA2, which is directly involved in transcriptional activation of ONSEN, was not higher in the drd1 mutant than in the wild-type. Interestingly, no transgenerational transposition of ONSEN was observed in the drd1 mutant, even though DNA methylation levels were significantly reduced and expression levels were increased compared to the wild-type. These results suggest that other factors are involved in the regulation of ONSEN transposition in addition to the transcript level of ONSEN.
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Affiliation(s)
| | - Yui Hayashi
- Graduate School of Life Science, Hokkaido University
| | - Kosuke Nozawa
- Graduate School of Life Science, Hokkaido University
| | - Lu Chen
- Graduate School of Life Science, Hokkaido University
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31
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Rizvanovic A, Kjellin J, Söderbom F, Holmqvist E. Saturation mutagenesis charts the functional landscape of Salmonella ProQ and reveals a gene regulatory function of its C-terminal domain. Nucleic Acids Res 2021; 49:9992-10006. [PMID: 34450657 PMCID: PMC8464044 DOI: 10.1093/nar/gkab721] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Revised: 08/03/2021] [Accepted: 08/10/2021] [Indexed: 01/14/2023] Open
Abstract
The global RNA-binding protein ProQ has emerged as a central player in post-transcriptional regulatory networks in bacteria. While the N-terminal domain (NTD) of ProQ harbors the major RNA-binding activity, the role of the ProQ C-terminal domain (CTD) has remained unclear. Here, we have applied saturation mutagenesis coupled to phenotypic sorting and long-read sequencing to chart the regulatory capacity of Salmonella ProQ. Parallel monitoring of thousands of ProQ mutants allowed mapping of critical residues in both the NTD and the CTD, while the linker separating these domains was tolerant to mutations. Single amino acid substitutions in the NTD associated with abolished regulatory capacity strongly align with RNA-binding deficiency. An observed cellular instability of ProQ associated with mutations in the NTD suggests that interaction with RNA protects ProQ from degradation. Mutation of conserved CTD residues led to overstabilization of RNA targets and rendered ProQ inert in regulation, without affecting protein stability in vivo. Furthermore, ProQ lacking the CTD, although binding competent, failed to protect an mRNA target from degradation. Together, our data provide a comprehensive overview of residues important for ProQ-dependent regulation and reveal an essential role for the enigmatic ProQ CTD in gene regulation.
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Affiliation(s)
- Alisa Rizvanovic
- Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Uppsala, S-75124, Sweden
| | - Jonas Kjellin
- Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Uppsala, S-75124, Sweden
| | - Fredrik Söderbom
- Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Uppsala, S-75124, Sweden
| | - Erik Holmqvist
- Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Uppsala, S-75124, Sweden
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32
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Ali Khan I. Do second generation sequencing techniques identify documented genetic markers for neonatal diabetes mellitus? Heliyon 2021; 7:e07903. [PMID: 34584998 PMCID: PMC8455689 DOI: 10.1016/j.heliyon.2021.e07903] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2020] [Revised: 01/15/2021] [Accepted: 08/27/2021] [Indexed: 12/24/2022] Open
Abstract
Neonatal diabetes mellitus (NDM) is noted as a genetic, heterogeneous, and rare disease in infants. NDM occurs due to a single-gene mutation in neonates. A common source for developing NDM in an infant is the existence of mutations/variants in the KCNJ11 and ABCC8 genes, encoding the subunits of the voltage-dependent potassium channel. Both KCNJ11 and ABCC8 genes are useful in diagnosing monogenic diabetes during infancy. Genetic analysis was previously performed using first-generation sequencing techniques, such as DNA-Sanger sequencing, which uses chain-terminating inhibitors. Sanger sequencing has certain limitations; it can screen a limited region of exons in one gene, but it cannot screen large regions of the human genome. In the last decade, first generation sequencing techniques have been replaced with second-generation sequencing techniques, such as next-generation sequencing (NGS), which sequences nucleic-acids more rapidly and economically than Sanger sequencing. NGS applications are involved in whole exome sequencing (WES), whole genome sequencing (WGS), and targeted gene panels. WES characterizes a substantial breakthrough in human genetics. Genetic testing for custom genes allows the screening of the complete gene, including introns and exons. The aim of this review was to confirm if the 22 genetic variations previously documented to cause NDM by Sanger sequencing could be detected using second generation sequencing techniques. The author has cross-checked global studies performed in NDM using NGS, ES/WES, WGS, and targeted gene panels as second-generation sequencing techniques; WES confirmed the similar variants, which have been previously documented with Sanger sequencing. WES is documented as a powerful tool and WGS as the most comprehensive test for verified the documented variants, as well as novel enhancers. This review recommends for the future studies should be performed with second generation sequencing techniques to identify the verified 22 genetic and novel variants by screening in NDM (PNDM or TNMD) children.
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Affiliation(s)
- Imran Ali Khan
- Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, PO Box-10219, Riyadh, 11433, Saudi Arabia
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33
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Ferraz RAC, Lopes ALG, da Silva JAF, Moreira DFV, Ferreira MJN, de Almeida Coimbra SV. DNA-protein interaction studies: a historical and comparative analysis. PLANT METHODS 2021; 17:82. [PMID: 34301293 PMCID: PMC8299673 DOI: 10.1186/s13007-021-00780-z] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/21/2021] [Accepted: 07/11/2021] [Indexed: 05/05/2023]
Abstract
DNA-protein interactions are essential for several molecular and cellular mechanisms, such as transcription, transcriptional regulation, DNA modifications, among others. For many decades scientists tried to unravel how DNA links to proteins, forming complex and vital interactions. However, the high number of techniques developed for the study of these interactions made the choice of the appropriate technique a difficult task. This review intends to provide a historical context and compile the methods that describe DNA-protein interactions according to the purpose of each approach, summarise the respective advantages and disadvantages and give some examples of recent uses for each technique. The final aim of this work is to help in deciding which technique to perform according to the objectives and capacities of each research team. Considering the DNA-binding proteins characterisation, filter binding assay and EMSA are easy in vitro methods that rapidly identify nucleic acid-protein binding interactions. To find DNA-binding sites, DNA-footprinting is indeed an easier, faster and reliable approach, however, techniques involving base analogues and base-site selection are more precise. Concerning binding kinetics and affinities, filter binding assay and EMSA are useful and easy methods, although SPR and spectroscopy techniques are more sensitive. Finally, relatively to genome-wide studies, ChIP-seq is the desired method, given the coverage and resolution of the technique. In conclusion, although some experiments are easier and faster than others, when designing a DNA-protein interaction study several concerns should be taken and different techniques may need to be considered, since different methods confer different precisions and accuracies.
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Affiliation(s)
- Ricardo André Campos Ferraz
- Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal
- LAQV Requimte, Sustainable Chemistry, Universidade do Porto, Porto, Portugal
| | - Ana Lúcia Gonçalves Lopes
- Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal
- LAQV Requimte, Sustainable Chemistry, Universidade do Porto, Porto, Portugal
| | - Jessy Ariana Faria da Silva
- LAQV Requimte, Sustainable Chemistry, Universidade do Porto, Porto, Portugal
- Universidade do Minho, Braga, Portugal
| | - Diana Filipa Viana Moreira
- Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal
- LAQV Requimte, Sustainable Chemistry, Universidade do Porto, Porto, Portugal
| | - Maria João Nogueira Ferreira
- Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal
- LAQV Requimte, Sustainable Chemistry, Universidade do Porto, Porto, Portugal
| | - Sílvia Vieira de Almeida Coimbra
- Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal.
- LAQV Requimte, Sustainable Chemistry, Universidade do Porto, Porto, Portugal.
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Green MR, Sambrook J. Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes. Cold Spring Harb Protoc 2021; 2021:2021/7/pdb.prot100495. [PMID: 34210770 DOI: 10.1101/pdb.prot100495] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
In this protocol, restriction fragments that have been transferred to a membrane by Southern blotting are hybridized to a labeled probe. Methods for stripping the probe from the membrane are also included.
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Abstract
In Southern blotting, DNA is digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through a standard agarose gel. The DNA is then denatured in situ and transferred from the gel to a solid support (usually a nylon or nitrocellulose membrane). The relative positions of the DNA fragments are preserved during their transfer to the membrane. The DNA is then fixed to the membrane and prepared for hybridization. Alternatively, DNA can be simultaneously transferred from the top and bottom surfaces of a single agarose gel to two membranes. This procedure is useful when the need arises to analyze the same set of restriction fragments with two different probes. Transfer of DNA fragments is rapid, but the efficiency is low because the agarose gel quickly becomes dehydrated as fluid is withdrawn from both sides. The method therefore works best when the target sequences are present in high concentration (e.g., when analyzing cloned DNAs [plasmids, bacteriophages, cosmids, PACs, or BACs] or less complex genomes [those of Saccharomyces cerevisiae or Drosophila]). Too little mammalian genomic DNA is transferred to allow signals from single-copy sequences to be detected in a reproducible or timely fashion.
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Green MR, Sambrook J. Analysis of DNA by Southern Blotting. Cold Spring Harb Protoc 2021; 2021:2021/7/pdb.top100396. [PMID: 34210774 DOI: 10.1101/pdb.top100396] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Southern transfer and hybridization are used to study how genes are organized within genomes by mapping restriction sites in and around segments of genomic DNA for which specific probes are available. Genomic DNA is first digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through a standard agarose gel. The DNA is then denatured in situ and transferred from the gel to a solid support (usually a nylon or nitrocellulose membrane). The DNA attached to the membrane is hybridized to a labeled DNA, RNA, or oligonucleotide probe, and bands complementary to the probe are located by an appropriate detection system (e.g., by autoradiography). By estimating the size and number of the bands generated after digestion of the genomic DNA with different restriction enzymes, singly or in combination, it is possible to place the target DNA within a context of restriction sites.
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Ahmad W, Gull B, Baby J, Mustafa F. A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach. Curr Issues Mol Biol 2021; 43:457-484. [PMID: 34206608 PMCID: PMC8929067 DOI: 10.3390/cimb43020036] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2021] [Revised: 06/07/2021] [Accepted: 06/16/2021] [Indexed: 12/27/2022] Open
Abstract
Northern blotting (NB), a gold standard for RNA detection, has lost its charm due to its hands-on nature, need for good quality RNA, and radioactivity. With the emergence of the field of microRNAs (miRNAs), the necessity for sensitive and quantitative NBs has again emerged. Here, we developed highly sensitive yet non-radiolabeled, fast, economical NB, and liquid hybridization (LH) assays without radioactivity or specialized reagents like locked nucleic acid (LNA)- or digoxigenin-labeled probes for mRNAs/small RNAs, especially miRNAs using biotinylated probes. An improvised means of hybridizing oligo probes along with efficient transfer, cross-linking, and signal enhancement techniques was employed. Important caveats of each assay were elaborated upon, especially issues related to probe biotinylation, use of exonuclease, and bioimagers not reported earlier. We demonstrate that, while the NBs were sensitive for mRNAs and small RNAs, our LH protocol could efficiently detect these and miRNAs using less than 10-100 times the total amount of RNA, a sensitivity comparable to radiolabeled probes. Compared to NBs, LH was a faster, more sensitive, and specific approach for mRNA/small RNA/miRNA detection. A comparison of present work with six seminal studies is presented along with detailed protocols for easy reproducibility. Overall, our study provides effective platforms to study large and small RNAs in a sensitive, efficient, and cost-effective manner.
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Lürig MD, Donoughe S, Svensson EI, Porto A, Tsuboi M. Computer Vision, Machine Learning, and the Promise of Phenomics in Ecology and Evolutionary Biology. Front Ecol Evol 2021. [DOI: 10.3389/fevo.2021.642774] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
For centuries, ecologists and evolutionary biologists have used images such as drawings, paintings and photographs to record and quantify the shapes and patterns of life. With the advent of digital imaging, biologists continue to collect image data at an ever-increasing rate. This immense body of data provides insight into a wide range of biological phenomena, including phenotypic diversity, population dynamics, mechanisms of divergence and adaptation, and evolutionary change. However, the rate of image acquisition frequently outpaces our capacity to manually extract meaningful information from images. Moreover, manual image analysis is low-throughput, difficult to reproduce, and typically measures only a few traits at a time. This has proven to be an impediment to the growing field of phenomics – the study of many phenotypic dimensions together. Computer vision (CV), the automated extraction and processing of information from digital images, provides the opportunity to alleviate this longstanding analytical bottleneck. In this review, we illustrate the capabilities of CV as an efficient and comprehensive method to collect phenomic data in ecological and evolutionary research. First, we briefly review phenomics, arguing that ecologists and evolutionary biologists can effectively capture phenomic-level data by taking pictures and analyzing them using CV. Next we describe the primary types of image-based data, review CV approaches for extracting them (including techniques that entail machine learning and others that do not), and identify the most common hurdles and pitfalls. Finally, we highlight recent successful implementations and promising future applications of CV in the study of phenotypes. In anticipation that CV will become a basic component of the biologist’s toolkit, our review is intended as an entry point for ecologists and evolutionary biologists that are interested in extracting phenotypic information from digital images.
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Pham N, Yan Z, Yu Y, Faria Afreen M, Malkova A, Haber JE, Ira G. Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks. EMBO J 2021; 40:e104847. [PMID: 33844333 DOI: 10.15252/embj.2020104847] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 03/04/2021] [Accepted: 03/09/2021] [Indexed: 11/09/2022] Open
Abstract
DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two-ended DNA double-strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single-ended DSBs are repaired by break-induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two-ended DSBs. Here, we demonstrate that BIR is suppressed at two-ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D-loop unwinding helicase Mph1, and (iii) Mre11-Rad50-Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans.
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Affiliation(s)
- Nhung Pham
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Zhenxin Yan
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Yang Yu
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Mosammat Faria Afreen
- Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA
| | - Anna Malkova
- Department of Biology, University of Iowa, Iowa City, IA, USA
| | - James E Haber
- Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA
| | - Grzegorz Ira
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
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Tripuraneni V, Memisoglu G, MacAlpine HK, Tran TQ, Zhu W, Hartemink AJ, Haber JE, MacAlpine DM. Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair in the absence of DNA replication. Genome Res 2021; 31:775-788. [PMID: 33811083 PMCID: PMC8092003 DOI: 10.1101/gr.271155.120] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Accepted: 03/26/2021] [Indexed: 12/27/2022]
Abstract
We interrogated at nucleotide resolution the spatiotemporal order of chromatin changes that occur immediately following a site-specific double-strand break (DSB) upstream of the PHO5 locus and its subsequent repair by nonhomologous end joining (NHEJ). We observed the immediate eviction of a nucleosome flanking the break and the repositioning of adjacent nucleosomes away from the break. These early chromatin events were independent of the end-processing Mre11-Rad50-Xrs2 (MRX) complex and preceded the MRX-dependent broad eviction of histones and DNA end-resectioning that extends up to ∼8 kb away from the break. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population. Concomitant with DSB repair by NHEJ, we observed the redeposition and precise repositioning of nucleosomes at their originally occupied positions. This re-establishment of the prelesion chromatin landscape suggests that a DNA replication-independent mechanism exists to preserve epigenome organization following DSB repair.
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Affiliation(s)
- Vinay Tripuraneni
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA
| | - Gonen Memisoglu
- Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA
| | - Heather K MacAlpine
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA
| | - Trung Q Tran
- Department of Computer Science, Duke University, Durham, North Carolina 27708, USA
| | - Wei Zhu
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA
| | | | - James E Haber
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454, USA
| | - David M MacAlpine
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA
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Potla P, Ali SA, Kapoor M. A bioinformatics approach to microRNA-sequencing analysis. OSTEOARTHRITIS AND CARTILAGE OPEN 2021; 3:100131. [DOI: 10.1016/j.ocarto.2020.100131] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2020] [Accepted: 12/14/2020] [Indexed: 01/20/2023] Open
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Bente H, Foerster AM, Lettner N, Mittelsten Scheid O. Polyploidy-associated paramutation in Arabidopsis is determined by small RNAs, temperature, and allele structure. PLoS Genet 2021; 17:e1009444. [PMID: 33690630 PMCID: PMC7978347 DOI: 10.1371/journal.pgen.1009444] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2020] [Revised: 03/19/2021] [Accepted: 02/24/2021] [Indexed: 11/18/2022] Open
Abstract
Paramutation is a form of non-Mendelian inheritance in which the expression of a paramutable allele changes when it encounters a paramutagenic allele. This change in expression of the paramutable alleles is stably inherited even after segregation of both alleles. While the discovery of paramutation and studies of its underlying mechanism were made with alleles that change plant pigmentation, paramutation-like phenomena are known to modulate the expression of other traits and in other eukaryotes, and many cases have probably gone undetected. It is likely that epigenetic mechanisms are responsible for the phenomenon, as paramutation forms epialleles, genes with identical sequences but different expression states. This could account for the intergenerational inheritance of the paramutated allele, providing profound evidence that triggered epigenetic changes can be maintained over generations. Here, we use a case of paramutation that affects a transgenic selection reporter gene in tetraploid Arabidopsis thaliana. Our data suggest that different types of small RNA are derived from paramutable and paramutagenic epialleles. In addition, deletion of a repeat within the epiallele changes its paramutability. Further, the temperature during the growth of the epiallelic hybrids determines the degree and timing of the allelic interaction. The data further make it plausible why paramutation in this system becomes evident only in the segregating F2 population of tetraploid plants containing both epialleles. In summary, the results support a model for polyploidy-associated paramutation, with similarities as well as distinctions from other cases of paramutation.
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Affiliation(s)
- Heinrich Bente
- Gregor Mendel Institute of Molecular Plant Biology (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria
| | - Andrea M. Foerster
- Gregor Mendel Institute of Molecular Plant Biology (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria
| | - Nicole Lettner
- Gregor Mendel Institute of Molecular Plant Biology (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria
| | - Ortrun Mittelsten Scheid
- Gregor Mendel Institute of Molecular Plant Biology (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria
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43
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Esmaeili N, Cai Y, Tang F, Zhu X, Smith J, Mishra N, Hequet E, Ritchie G, Jones D, Shen G, Payton P, Zhang H. Towards doubling fibre yield for cotton in the semiarid agricultural area by increasing tolerance to drought, heat and salinity simultaneously. PLANT BIOTECHNOLOGY JOURNAL 2021; 19:462-476. [PMID: 32902115 PMCID: PMC7955890 DOI: 10.1111/pbi.13476] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/23/2020] [Revised: 08/19/2020] [Accepted: 08/23/2020] [Indexed: 05/15/2023]
Abstract
Abiotic stresses such as extreme temperatures, water-deficit and salinity negatively affect plant growth and development, and cause significant yield losses. It was previously shown that co-overexpression of the Arabidopsis vacuolar pyrophosphatase gene AVP1 and the rice SUMO E3 ligase gene OsSIZ1 in Arabidopsis significantly increased tolerance to multiple abiotic stresses and led to increased seed yield for plants grown under single or multiple abiotic stress conditions. It was hypothesized that there might be synergistic effects between AVP1 overexpression and OsSIZ1 overexpression, which could lead to substantially increased yields if these two genes are co-overexpressed in real crops. To test this hypothesis, AVP1 and OsSIZ1 were co-overexpressed in cotton, and the impact of OsSIZ1/AVP1 co-overexpression on cotton's performance under normal growth and multiple stress conditions were analysed. It was found that OsSIZ1/AVP1 co-overexpressing plants performed significantly better than AVP1-overexpressing, OsSIZ1-overexpressing and wild-type cotton plants under single, as well as under multiple stress conditions in laboratory and field conditions. Two field studies showed that OsSIZ1/AVP1 co-overexpressing plants produced 133% and 81% more fibre than wild-type cotton in the dryland conditions of West Texas. This research illustrates that co-overexpression of AVP1 and OsSIZ1 is a viable strategy for engineering abiotic stress-tolerant crops and could substantially improve crop yields in low input or marginal environments, providing a solution for food security for countries in arid and semiarid regions of the world.
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Affiliation(s)
- Nardana Esmaeili
- Department of Biological SciencesTexas Tech UniversityLubbockTXUSA
| | - Yifan Cai
- Department of Biological SciencesTexas Tech UniversityLubbockTXUSA
| | - Feiyu Tang
- College of AgronomyJiangxi Agricultural UniversityNanchangChina
| | - Xunlu Zhu
- Department of Biological SciencesTexas Tech UniversityLubbockTXUSA
| | - Jennifer Smith
- Department of Biological SciencesTexas Tech UniversityLubbockTXUSA
| | - Neelam Mishra
- St. Joseph's College AutonomousBengaluruKarnatakaIndia
| | - Eric Hequet
- Department of Plant and Soil ScienceTexas Tech UniversityLubbockTXUSA
| | - Glen Ritchie
- Department of Plant and Soil ScienceTexas Tech UniversityLubbockTXUSA
| | | | - Guoxin Shen
- Zhejiang Academy of Agricultural SciencesHangzhouChina
| | - Paxton Payton
- USDA‐ARS Cropping Systems Research LaboratoryLubbockTXUSA
| | - Hong Zhang
- Department of Biological SciencesTexas Tech UniversityLubbockTXUSA
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Yu H, Kong X, Huang H, Wu W, Park J, Yun DJ, Lee BH, Shi H, Zhu JK. STCH4/REIL2 Confers Cold Stress Tolerance in Arabidopsis by Promoting rRNA Processing and CBF Protein Translation. Cell Rep 2021; 30:229-242.e5. [PMID: 31914389 DOI: 10.1016/j.celrep.2019.12.012] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2019] [Revised: 10/29/2019] [Accepted: 12/04/2019] [Indexed: 12/20/2022] Open
Abstract
Plants respond to cold stress by inducing the expression of transcription factors that regulate downstream genes to confer tolerance to freezing. We screened an Arabidopsis transfer DNA (T-DNA) insertion library and identified a cold-hypersensitive mutant, which we named stch4 (sensitive to chilling 4). STCH4/REIL2 encodes a ribosomal biogenesis factor that is upregulated upon cold stress. Overexpression of STCH4 confers chilling and freezing tolerance in Arabidopsis. The stch4 mutation reduces CBF protein levels and thus delayed the induction of C-repeat-binding factor (CBF) regulon genes. Ribosomal RNA processing is reduced in stch4 mutants, especially under cold stress. STCH4 associates with multiple ribosomal proteins, and these interactions are modulated by cold stress. These results suggest that the ribosome is a regulatory node for cold stress responses and that STCH4 promotes an altered ribosomal composition and functions in low temperatures to facilitate the translation of proteins important for plant growth and survival under cold stress.
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Affiliation(s)
- Hasi Yu
- Shanghai Center for Plant Stress Biology, Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China; Institute of Plant Physiology and Ecology, Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China; University of Chinese Academy of Sciences, Shanghai 200032, People's Republic of China
| | - Xiangfeng Kong
- Shanghai Center for Plant Stress Biology, Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China; University of Chinese Academy of Sciences, Shanghai 200032, People's Republic of China
| | - Huan Huang
- Shanghai Center for Plant Stress Biology, Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China; University of Chinese Academy of Sciences, Shanghai 200032, People's Republic of China
| | - Wenwu Wu
- The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, 311300 Lin'an, Hangzhou, People's Republic of China
| | - Junghoon Park
- Department of Biomedical Science and Engineering, Konkuk University, Seoul 05029, South Korea
| | - Dae-Jin Yun
- Department of Biomedical Science and Engineering, Konkuk University, Seoul 05029, South Korea
| | - Byeong-Ha Lee
- Department of Life Science, Sogang University, Seoul 04107, South Korea
| | - Huazhong Shi
- Shanghai Center for Plant Stress Biology, Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China; Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA.
| | - Jian-Kang Zhu
- Shanghai Center for Plant Stress Biology, Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China; Institute of Plant Physiology and Ecology, Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China; Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA.
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Abstract
RNA samples that have been transferred and fixed to a membrane may be hybridized with a specific probe to locate the RNA species of interest. Any one of a large number of methods can be used to label and detect probes, at the discretion of the investigator. After treating the membrane with blocking agents that suppress nonspecific absorption of the probe, the membrane is incubated under conditions that favor hybridization of the labeled probe to the immobilized target RNA. The membrane is then washed extensively to remove adventitiously bound probe and finally manipulated to yield an image of the distribution of the tightly bound probe on the membrane. After analysis of the results, the probe may be stripped from the membrane, and the membrane can then be used again in another hybridization experiment.
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46
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Abstract
A number of transposable elements are activated by environmental stress. A Ty1/copia-type retrotransposon named ONSEN is activated by heat stress in Brassicaceae species. A synthetic activation of the transposon is effective for the molecular breeding without genetic modification. Here, we described the detail procedure of heat treatment to activate ONSEN in Brassicaceae species.
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Affiliation(s)
- Hidetaka Ito
- Faculty of Science, Hokkaido University, Sapporo, Hokkaido, Japan.
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47
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Osia B, Elango R, Kramara J, Roberts SA, Malkova A. Investigation of Break-Induced Replication in Yeast. Methods Mol Biol 2021; 2153:307-328. [PMID: 32840789 PMCID: PMC9041317 DOI: 10.1007/978-1-0716-0644-5_22] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/02/2023]
Abstract
Repair of double-strand DNA breaks (DSBs) is important for preserving genomic integrity and stability. Break-induced replication (BIR) is a mechanism aimed to repair one-ended double-strand DNA breaks, similar to those formed by replication fork collapse or by telomere erosion. Unlike S-phase replication, BIR is carried out by a migrating DNA bubble and is associated with conservative inheritance of newly synthesized DNA. This unusual DNA synthesis leads to high level of mutagenesis and chromosomal rearrangements during BIR. Here, we focus on several genetic and molecular methods to investigate BIR using our system in yeast Saccharomyces cerevisiae where BIR is initiated by a site-specific DNA break, and the repair involves two copies of chromosome III.
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Affiliation(s)
- Beth Osia
- Department of Biology, University of Iowa, Iowa City, IA, USA
| | - Rajula Elango
- Department of Medicine, Division of Hematology-Oncology, Cancer Research Institute, Harvard Medical School, Boston, MA, USA
- Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Juraj Kramara
- Department of Biology, University of Iowa, Iowa City, IA, USA
| | - Steven A Roberts
- School of Molecular Biosciences, Center for Reproductive Biology, Washington State University, Pullman, WA, USA
| | - Anna Malkova
- Department of Biology, University of Iowa, Iowa City, IA, USA.
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The Small Toxic Salmonella Protein TimP Targets the Cytoplasmic Membrane and Is Repressed by the Small RNA TimR. mBio 2020; 11:mBio.01659-20. [PMID: 33172998 PMCID: PMC7667032 DOI: 10.1128/mbio.01659-20] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life. To reduce complexity when new genome sequences are annotated, open reading frames (ORFs) shorter than 50 codons in length are generally omitted. However, it has recently become evident that this procedure sorts away ORFs encoding small proteins of high biological significance. For instance, tailored small protein identification approaches have shown that bacteria encode numerous small proteins with important physiological functions. As the number of predicted small ORFs increase, it becomes important to characterize the corresponding proteins. In this study, we discovered a conserved but previously overlooked small enterobacterial protein. We show that this protein, which we dubbed TimP, is a potent toxin that inhibits bacterial growth by targeting the cell membrane. Toxicity is relieved by a small regulatory RNA, which binds the toxin mRNA to inhibit toxin synthesis. Small proteins are gaining increased attention due to their important functions in major biological processes throughout the domains of life. However, their small size and low sequence conservation make them difficult to identify. It is therefore not surprising that enterobacterial ryfA has escaped identification as a small protein coding gene for nearly 2 decades. Since its identification in 2001, ryfA has been thought to encode a noncoding RNA and has been implicated in biofilm formation in Escherichia coli and pathogenesis in Shigella dysenteriae. Although a recent ribosome profiling study suggested ryfA to be translated, the corresponding protein product was not detected. In this study, we provide evidence that ryfA encodes a small toxic inner membrane protein, TimP, overexpression of which causes cytoplasmic membrane leakage. TimP carries an N-terminal signal sequence, indicating that its membrane localization is Sec-dependent. Expression of TimP is repressed by the small RNA (sRNA) TimR, which base pairs with the timP mRNA to inhibit its translation. In contrast to overexpression, endogenous expression of TimP upon timR deletion permits cell growth, possibly indicating a toxicity-independent function in the bacterial membrane.
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Boonjing P, Masuta Y, Nozawa K, Kato A, Ito H. The effect of zebularine on the heat-activated retrotransposon ONSEN in Arabidopsis thaliana and Vigna angularis. Genes Genet Syst 2020; 95:165-172. [PMID: 32741853 DOI: 10.1266/ggs.19-00046] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
The Ty1/copia-like retrotransposon ONSEN is conserved among Brassica species, as well as in beans, including adzuki bean (Vigna angularis (Willd.) Ohwi & Ohashi), which is one of the economically important crops in Japan. ONSEN has acquired a heat-responsive element that is recognized by plant heat stress defense factors, resulting in its transcription and the production of full-length extrachromosomal DNA under conditions with elevated temperatures. DNA methylation plays an important role in regulating the activation of this transposon in plants. Therefore, chemical inhibition of DNA methyltransferases has been utilized to study the effect of DNA methylation on transposon activation. To understand the effect of DNA methylation on ONSEN activation, Arabidopsis thaliana and adzuki bean seedlings were treated with zebularine, which is known to be an effective chemical demethylation agent. The results showed that ONSEN transcription levels were upregulated in zebularine-treated plants. Extrachromosomal DNA of ONSEN also accumulated in the treated plants.
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Affiliation(s)
| | | | - Kosuke Nozawa
- Graduate School of Life Science, Hokkaido University
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Li S, Tollefsbol TO. DNA methylation methods: Global DNA methylation and methylomic analyses. Methods 2020; 187:28-43. [PMID: 33039572 DOI: 10.1016/j.ymeth.2020.10.002] [Citation(s) in RCA: 107] [Impact Index Per Article: 21.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2020] [Revised: 10/02/2020] [Accepted: 10/05/2020] [Indexed: 12/13/2022] Open
Abstract
DNA methylation provides a pivotal layer of epigenetic regulation in eukaryotes that has significant involvement for numerous biological processes in health and disease. The function of methylation of cytosine bases in DNA was originally proposed as a "silencing" epigenetic marker and focused on promoter regions of genes for decades. Improved technologies and accumulating studies have been extending our understanding of the roles of DNA methylation to various genomic contexts including gene bodies, repeat sequences and transcriptional start sites. The demand for comprehensively describing DNA methylation patterns spawns a diversity of DNA methylation profiling technologies that target its genomic distribution. These approaches have enabled the measurement of cytosine methylation from specific loci at restricted regions to single-base-pair resolution on a genome-scale level. In this review, we discuss the different DNA methylation analysis technologies primarily based on the initial treatments of DNA samples: bisulfite conversion, endonuclease digestion and affinity enrichment, involving methodology evolution, principles, applications, and their relative merits. This review may offer referable information for the selection of various platforms for genome-wide analysis of DNA methylation.
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Affiliation(s)
- Shizhao Li
- Department of Biology, University of Alabama at Birmingham, Birmingham, AL, United States.
| | - Trygve O Tollefsbol
- Department of Biology, University of Alabama at Birmingham, Birmingham, AL, United States; Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, United States; Nutrition Obesity Research Center, University of Alabama at Birmingham, Birmingham, AL, United States; Comprehensive Center for Healthy Aging, University of Alabama at Birmingham, Birmingham, AL, United States; Comprehensive Diabetes Center, University of Alabama at Birmingham, Birmingham, AL, United States.
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