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Gál L, Schamberger A, Wachtl G, Orbán TI. The Effect of Alternative Splicing Sites on Mirtron Formation and Arm Selection of Precursor microRNAs. Int J Mol Sci 2024; 25:7643. [PMID: 39062888 PMCID: PMC11277307 DOI: 10.3390/ijms25147643] [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: 05/13/2024] [Revised: 06/25/2024] [Accepted: 07/08/2024] [Indexed: 07/28/2024] Open
Abstract
Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery for their maturation. However, the molecular details of this Drosha-independent processing are still not fully understood; as an example, the Microprocessor complex cannot process the mirtronic pre-miRNA from the transcript even if splice site mutations are present. To investigate the influence of alternative splicing sites on mirtron formation, we generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial introns to compare the processing of canonical miRNAs and mirtrons. Although mutations of both splice sites generated a complex pattern of alternative transcripts, mirtron formation was always severely affected as opposed to the normal processing of the canonical hsa-mir-33b miRNA. However, we also detected that while its formation was also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that this phenomenon is not dependent on Microprocessor activity but rather points toward the potential stability difference between the miRNAs from the different arms. Our results indicate that when the major splice sites are mutated, mirtron formation cannot be rescued by nearby alternative splice sites, and stability differences between 5p and 3p species should also be considered for functional studies of mirtrons.
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Affiliation(s)
- Luca Gál
- Gene Regulation Research Group, Institute of Molecular Life Sciences, HUN-REN Research Centre for Natural Sciences, 1117 Budapest, Hungary
- Doctoral School of Biology, Institute of Biology, ELTE Eötvös Loránd University, 1117 Budapest, Hungary
| | - Anita Schamberger
- Gene Regulation Research Group, Institute of Molecular Life Sciences, HUN-REN Research Centre for Natural Sciences, 1117 Budapest, Hungary
- Institute of Genetics and Biotechnology, Hungarian University of Agriculture and Life Sciences, 2100 Gödöllő, Hungary
| | - Gerda Wachtl
- Gene Regulation Research Group, Institute of Molecular Life Sciences, HUN-REN Research Centre for Natural Sciences, 1117 Budapest, Hungary
| | - Tamás I. Orbán
- Gene Regulation Research Group, Institute of Molecular Life Sciences, HUN-REN Research Centre for Natural Sciences, 1117 Budapest, Hungary
- Doctoral School of Biology, Institute of Biology, ELTE Eötvös Loránd University, 1117 Budapest, Hungary
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2
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Orbán TI. One locus, several functional RNAs-emerging roles of the mechanisms responsible for the sequence variability of microRNAs. Biol Futur 2023:10.1007/s42977-023-00154-7. [PMID: 36847925 DOI: 10.1007/s42977-023-00154-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2022] [Accepted: 02/08/2023] [Indexed: 03/01/2023]
Abstract
With the development of modern molecular genetics, the original "one gene-one enzyme" hypothesis has been outdated. For protein coding genes, the discovery of alternative splicing and RNA editing provided the biochemical background for the RNA repertoire of a single locus, which also serves as an important pillar for the enormous protein variability of the genomes. Non-protein coding RNA genes were also revealed to produce several RNA species with distinct functions. The loci of microRNAs (miRNAs), encoding for small endogenous regulatory RNAs, were also found to produce a population of small RNAs, rather than a single defined product. This review aims to present the mechanisms contributing to the astonishing variability of miRNAs revealed by the new sequencing technologies. One important source is the careful balance of arm selection, producing sequentially different 5p- or 3p-miRNAs from the same pre-miRNA, thereby broadening the number of regulated target RNAs and the phenotypic response. In addition, the formation of 5', 3' and polymorphic isomiRs, with variable end and internal sequences also leads to a higher number of targeted sequences, and increases the regulatory output. These miRNA maturation processes, together with other known mechanisms such as RNA editing, further increase the potential outcome of this small RNA pathway. By discussing the subtle mechanisms behind the sequence diversity of miRNAs, this review intends to reveal this engaging aspect of the inherited "RNA world", how it contributes to the almost infinite molecular variability among living organisms, and how this variability can be exploited to treat human diseases.
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Affiliation(s)
- Tamás I Orbán
- Institute of Enzymology, Research Centre for Natural Sciences, Eötvös Loránd Research Network, Magyar Tudósok Körútja 2, Budapest, 1117, Hungary.
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3
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Partial Disturbance of Microprocessor Function in Human Stem Cells Carrying a Heterozygous Mutation in the DGCR8 Gene. Genes (Basel) 2022; 13:genes13111925. [DOI: 10.3390/genes13111925] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Revised: 10/14/2022] [Accepted: 10/20/2022] [Indexed: 11/04/2022] Open
Abstract
Maturation of microRNAs (miRNAs) begins by the “Microprocessor” complex, containing the Drosha endonuclease and its partner protein, "DiGeorge Syndrome Critical Region 8" (DGCR8). Although the main function of the two proteins is to coordinate the first step of precursor miRNAs formation, several studies revealed their miRNA-independent functions in other RNA-related pathways (e.g., in snoRNA decay) or, for the DGCR8, the role in tissue development. To investigate the specific roles of DGCR8 in various cellular pathways, we previously established a human embryonic stem-cell (hESC) line carrying a monoallelic DGCR8 mutation by using the CRISPR-Cas9 system. In this study, we genetically characterized single-cell originated progenies of the cell line and showed that DGCR8 heterozygous mutation results in only a modest effect on the mRNA level but a significant decrease at the protein level. Self-renewal and trilineage differentiation capacity of these hESCs were not affected by the mutation. However, partial disturbance of the Microprocessor function could be revealed in pri-miRNA processing along the human chromosome 19 miRNA cluster in several clones. With all these studies, we can demonstrate that the mutant hESC line is a good model to study not only miRNA-related but also other “noncanonical” functions of the DGCR8 protein.
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Zhou H, Hussain SS, Shi BJ. One vector-based method to verify predicted plant miRNAs, target sequences, and function modes. Biotechnol Bioeng 2021; 118:3105-3116. [PMID: 34002369 DOI: 10.1002/bit.27821] [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] [Received: 03/21/2021] [Revised: 05/02/2021] [Accepted: 05/12/2021] [Indexed: 11/09/2022]
Abstract
Many microRNAs (miRNAs) have been predicted from small RNA sequencing data, but little was experimentally verified due to the lack of effective methods. Here, we developed a simple and reliable dual gene expression cassette vector-based method to verify predicted plant miRNAs. We cloned osa-miR528 as a known miRNA, hvu-miRX as a predicted miRNA and TaDREB3 open reading frame as a non-miRNA into the first gene expression cassette and fused their complementary or noncomplementary sequences as predicted target or nontarget sequences with the 3' untranslated region of green fluorescent protein (GFP) in the second one. When these constructs were bombarded into plant cells, only the construct containing both osa-miR528 or hvu-miRX and its complementary sequence did not generate green fluorescence. Stem-loop reverse-transcription polymerase chain reaction detected mature osa-miR528 or mature hvu-miRX in the cells, while northern analysis showed that GFP messenger RNA from the construct containing both osa-miR528 or hvu-miRX and its complementary sequence was degraded. Taken together, the results indicate that hvu-miRX is an authentic miRNA like osa-miR528, miRNA's complementary sequence is its target sequence, and both osa-miR528 and hvu-miRX silenced the GFP expression via a cleavage mode. Our method thus facilitates the verification of predicted plant miRNAs, target sequences, and function modes.
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Affiliation(s)
- Hui Zhou
- School of Agriculture, Food and Wine, University of Adelaide, Urrbrae, South Australia, Australia
| | - Syed S Hussain
- School of Agriculture, Food and Wine, University of Adelaide, Urrbrae, South Australia, Australia
| | - Bu-Jun Shi
- School of Agriculture, Food and Wine, University of Adelaide, Urrbrae, South Australia, Australia
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5
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Posttranscriptional Regulation of the Human ABCG2 Multidrug Transporter Protein by Artificial Mirtrons. Genes (Basel) 2021; 12:genes12071068. [PMID: 34356084 PMCID: PMC8307164 DOI: 10.3390/genes12071068] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2021] [Revised: 07/08/2021] [Accepted: 07/09/2021] [Indexed: 12/24/2022] Open
Abstract
ABCG2 is a membrane transporter protein that has been associated with multidrug resistance phenotype and tumor development. Additionally, it is expressed in various stem cells, providing cellular protection against endobiotics and xenobiotics. In this study, we designed artificial mirtrons to regulate ABCG2 expression posttranscriptionally. Applying EGFP as a host gene, we could achieve efficient silencing not only in luciferase reporter systems but also at the ABCG2 protein level. Moreover, we observed important new sequential-functional features of the designed mirtrons. Mismatch at the first position of the mirtron-derived small RNA resulted in better silencing than full complementarity, while the investigated middle and 3′ mismatches did not enhance silencing. These latter small RNAs operated most probably via non-seed specific translational inhibition in luciferase assays. Additionally, we found that a mismatch in the first position has not, but a second mismatch in the third position has abolished target mRNA decay. Besides, one nucleotide mismatch in the seed region did not impair efficient silencing at the protein level, providing the possibility to silence targets carrying single nucleotide polymorphisms or mutations. Taken together, we believe that apart from establishing an efficient ABCG2 silencing system, our designing pipeline and results on sequential-functional features are beneficial for developing artificial mirtrons for other targets.
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Alles J, Fehlmann T, Fischer U, Backes C, Galata V, Minet M, Hart M, Abu-Halima M, Grässer FA, Lenhof HP, Keller A, Meese E. An estimate of the total number of true human miRNAs. Nucleic Acids Res 2019; 47:3353-3364. [PMID: 30820533 PMCID: PMC6468295 DOI: 10.1093/nar/gkz097] [Citation(s) in RCA: 376] [Impact Index Per Article: 62.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2018] [Revised: 01/30/2019] [Accepted: 02/07/2019] [Indexed: 02/06/2023] Open
Abstract
While the number of human miRNA candidates continuously increases, only a few of them are completely characterized and experimentally validated. Toward determining the total number of true miRNAs, we employed a combined in silico high- and experimental low-throughput validation strategy. We collected 28 866 human small RNA sequencing data sets containing 363.7 billion sequencing reads and excluded falsely annotated and low quality data. Our high-throughput analysis identified 65% of 24 127 mature miRNA candidates as likely false-positives. Using northern blotting, we experimentally validated miRBase entries and novel miRNA candidates. By exogenous overexpression of 108 precursors that encode 205 mature miRNAs, we confirmed 68.5% of the miRBase entries with the confirmation rate going up to 94.4% for the high-confidence entries and 18.3% of the novel miRNA candidates. Analyzing endogenous miRNAs, we verified the expression of 8 miRNAs in 12 different human cell lines. In total, we extrapolated 2300 true human mature miRNAs, 1115 of which are currently annotated in miRBase V22. The experimentally validated miRNAs will contribute to revising targetomes hypothesized by utilizing falsely annotated miRNAs.
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Affiliation(s)
- Julia Alles
- Institute of Human Genetics, Saarland University, 66421 Homburg, Germany
| | - Tobias Fehlmann
- Chair for Clinical Bioinformatics, Saarland University, 66123 Saarbrücken, Germany
| | - Ulrike Fischer
- Institute of Human Genetics, Saarland University, 66421 Homburg, Germany
| | - Christina Backes
- Chair for Clinical Bioinformatics, Saarland University, 66123 Saarbrücken, Germany
| | - Valentina Galata
- Chair for Clinical Bioinformatics, Saarland University, 66123 Saarbrücken, Germany
| | - Marie Minet
- Institute of Human Genetics, Saarland University, 66421 Homburg, Germany.,Chair for Clinical Bioinformatics, Saarland University, 66123 Saarbrücken, Germany
| | - Martin Hart
- Institute of Human Genetics, Saarland University, 66421 Homburg, Germany
| | - Masood Abu-Halima
- Institute of Human Genetics, Saarland University, 66421 Homburg, Germany
| | - Friedrich A Grässer
- Institute of Virology, Saarland University Medical School, 66421 Homburg, Germany
| | - Hans-Peter Lenhof
- Chair for Bioinformatics, Center for Bioinformatics, Saarland Informatics Campus, 66123 Saarbrücken, Germany
| | - Andreas Keller
- Chair for Clinical Bioinformatics, Saarland University, 66123 Saarbrücken, Germany
| | - Eckart Meese
- Institute of Human Genetics, Saarland University, 66421 Homburg, Germany
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Abstract
BACKGROUND MicroRNAs proceeds through the different canonical and non-canonical pathways; the most frequent of the non-canonical ones is the splicing-dependent biogenesis of mirtrons. We compare the mirtrons and non-mirtrons of human and mouse to explore how their maturation appears in the precursor structure around the miRNA. RESULTS We found the coherence of the overhang lengths what indicates the dependence between the cleavage sites. To explain this dependence we suggest the 2-lever model of the Dicer structure that couples the imprecisions in Drosha and Dicer. Considering the secondary structure of all animal pre-miRNAs we confirmed that single-stranded nucleotides tend to be located near the miRNA boundaries and in its center and are characterized by a higher mutation rate. The 5' end of the canonical 5' miRNA approaches the nearest single-stranded nucleotides what suggests the extension of the loop-counting rule from the Dicer to the Drosha cleavage site. A typical structure of the annotated mirtron pre-miRNAs differs from the canonical pre-miRNA structure and possesses the 1- and 2 nt hanging ends at the hairpin base. Together with the excessive variability of the mirtron Dicer cleavage site (that could be partially explained by guanine at its ends inherited from splicing) this is one more evidence for the 2-lever model. In contrast with the canonical miRNAs the mirtrons have higher snp densities and their pre-miRNAs are inversely associated with diseases. Therefore we supported the view that mirtrons are under positive selection while canonical miRNAs are under negative one and we suggested that mirtrons are an intrinsic source of silencing variability which produces the disease-promoting variants. Finally, we considered the interference of the pre-miRNA structure and the U2snRNA:pre-mRNA basepairing. We analyzed the location of the branchpoints and found that mirtron structure tends to expose the branchpoint site what suggests that the mirtrons can readily evolve from occasional hairpins in the immediate neighbourhood of the 3' splice site. CONCLUSION The miRNA biogenesis manifests itself in the footprints of the secondary structure. Close inspection of these structural properties can help to uncover new pathways of miRNA biogenesis and to refine the known miRNA data, in particular, new non-canonical miRNAs may be predicted or the known miRNAs can be re-classified.
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Affiliation(s)
- Igor I Titov
- Federal State Budget Scientific Institution "The Federal Research Center Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences", Novosibirsk, Russia. .,Novosibirsk State University, Novosibirsk, Russia.
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8
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Curtis HJ, Seow Y, Wood MJA, Varela MA. Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7. Nucleic Acids Res 2017; 45:7870-7885. [PMID: 28575281 PMCID: PMC5569705 DOI: 10.1093/nar/gkx483] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2016] [Accepted: 05/26/2017] [Indexed: 12/13/2022] Open
Abstract
We evaluate a knockdown-replacement strategy mediated by mirtrons as an alternative to allele-specific silencing using spinocerebellar ataxia 7 (SCA7) as a model. Mirtrons are introns that form pre-microRNA hairpins after splicing, producing RNAi effectors not processed by Drosha. Mirtron mimics may therefore avoid saturation of the canonical processing pathway. This method combines gene silencing mediated by an artificial mirtron with delivery of a functional copy of the gene such that both elements of the therapy are always expressed concurrently, minimizing the potential for undesirable effects and preserving wild-type function. This mutation- and single nucleotide polymorphism-independent method could be crucial in dominant diseases that feature both gain- and loss-of-function pathologies or have a heterogeneous genetic background. Here we develop mirtrons against ataxin 7 with silencing efficacy comparable to shRNAs, and introduce silent mutations into an ataxin 7 transgene such that it is resistant to their effect. We successfully express the transgene and one mirtron together from a single construct. Hence, we show that this method can be used to silence the endogenous allele of ataxin 7 and replace it with an exogenous copy of the gene, highlighting the efficacy and transferability across patient genotypes of this approach.
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Affiliation(s)
- Helen J Curtis
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK.,Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford OX2 6GG, UK
| | - Yiqi Seow
- Molecular Engineering Laboratory, Biomedical Sciences Institutes, A*STAR, Singapore
| | - Matthew J A Wood
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK
| | - Miguel A Varela
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK
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Drury RE, O'Connor D, Pollard AJ. The Clinical Application of MicroRNAs in Infectious Disease. Front Immunol 2017; 8:1182. [PMID: 28993774 PMCID: PMC5622146 DOI: 10.3389/fimmu.2017.01182] [Citation(s) in RCA: 121] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2017] [Accepted: 09/06/2017] [Indexed: 12/19/2022] Open
Abstract
MicroRNAs (miRNAs) are short single-stranded non-coding RNA sequences that posttranscriptionally regulate up to 60% of protein encoding genes. Evidence is emerging that miRNAs are key mediators of the host response to infection, predominantly by regulating proteins involved in innate and adaptive immune pathways. miRNAs can govern the cellular tropism of some viruses, are implicated in the resistance of some individuals to infections like HIV, and are associated with impaired vaccine response in older people. Not surprisingly, pathogens have evolved ways to undermine the effects of miRNAs on immunity. Recognition of this has led to new experimental treatments, RG-101 and Miravirsen—hepatitis C treatments which target host miRNA. miRNAs are being investigated as novel infection biomarkers, and they are being used to design attenuated vaccines, e.g., against Dengue virus. This comprehensive review synthesizes current knowledge of miRNA in host response to infection with emphasis on potential clinical applications, along with an evaluation of the challenges still to be overcome.
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Affiliation(s)
- Ruth E Drury
- Oxford Vaccine Group, Centre for Clinical Vaccinology and Tropical Medicine, Department of Paediatrics, University of Oxford, The Churchill Hospital, Oxford, United Kingdom
| | - Daniel O'Connor
- Oxford Vaccine Group, Centre for Clinical Vaccinology and Tropical Medicine, Department of Paediatrics, University of Oxford, The Churchill Hospital, Oxford, United Kingdom
| | - Andrew J Pollard
- Oxford Vaccine Group, Centre for Clinical Vaccinology and Tropical Medicine, Department of Paediatrics, University of Oxford, The Churchill Hospital, Oxford, United Kingdom
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Hubé F, Ulveling D, Sureau A, Forveille S, Francastel C. Short intron-derived ncRNAs. Nucleic Acids Res 2017; 45:4768-4781. [PMID: 28053119 PMCID: PMC5416886 DOI: 10.1093/nar/gkw1341] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2016] [Accepted: 12/21/2016] [Indexed: 01/02/2023] Open
Abstract
Introns represent almost half of the human genome, although they are eliminated from transcripts through RNA splicing. Yet, different classes of non-canonical miRNAs have been proposed to originate directly from intron splicing. Here, we considered the alternative splicing of introns as an interesting source of miRNAs, compatible with a developmental switch. We report computational prediction of new Short Intron-Derived ncRNAs (SID), defined as precursors of smaller ncRNAs like miRNAs and snoRNAs produced directly by splicing, and tested their dependence on each key factor in canonical or alternative miRNAs biogenesis (Drosha, DGCR8, DBR1, snRNP70, U2AF65, PRP8, Dicer, Ago2). We found that about half of predicted SID rely on debranching of the excised intron-lariat by the enzyme DBR1, as proposed for mirtrons. However, we identified new classes of SID for which miRNAs biogenesis may rely on intermingling between canonical and alternative pathways. We validated selected SID as putative miRNAs precursors and identified new endogenous miRNAs produced by non-canonical pathways, including one hosted in the first intron of SRA (Steroid Receptor RNA activator). Consistent with increased SRA intron retention during myogenic differentiation, release of SRA intron and its associated mature miRNA decreased in cells from healthy subjects but not from myotonic dystrophy patients with splicing defects.
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Affiliation(s)
- Florent Hubé
- Université Paris Diderot, Sorbonne Paris Cité, Paris, France.,Epigénétique et Destin Cellulaire, CNRS UMR 7216, Paris, France
| | - Damien Ulveling
- Université Paris Diderot, Sorbonne Paris Cité, Paris, France.,Epigénétique et Destin Cellulaire, CNRS UMR 7216, Paris, France
| | - Alain Sureau
- Université Paris Diderot, Sorbonne Paris Cité, Paris, France.,Epigénétique et Destin Cellulaire, CNRS UMR 7216, Paris, France
| | - Sabrina Forveille
- Université Paris Diderot, Sorbonne Paris Cité, Paris, France.,Epigénétique et Destin Cellulaire, CNRS UMR 7216, Paris, France
| | - Claire Francastel
- Université Paris Diderot, Sorbonne Paris Cité, Paris, France.,Epigénétique et Destin Cellulaire, CNRS UMR 7216, Paris, France
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Paces J, Nic M, Novotny T, Svoboda P. Literature review of baseline information to support the risk assessment of RNAi‐based GM plants. ACTA ACUST UNITED AC 2017. [PMCID: PMC7163844 DOI: 10.2903/sp.efsa.2017.en-1246] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Affiliation(s)
- Jan Paces
- Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic (IMG)
| | | | | | - Petr Svoboda
- Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic (IMG)
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Bonizzato A, Gaffo E, te Kronnie G, Bortoluzzi S. CircRNAs in hematopoiesis and hematological malignancies. Blood Cancer J 2016; 6:e483. [PMID: 27740630 PMCID: PMC5098259 DOI: 10.1038/bcj.2016.81] [Citation(s) in RCA: 123] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2016] [Accepted: 08/11/2016] [Indexed: 12/12/2022] Open
Abstract
Cell states in hematopoiesis are controlled by master regulators and by complex circuits of a growing family of RNA species impacting cell phenotype maintenance and plasticity. Circular RNAs (circRNAs) are rapidly gaining the status of particularly stable transcriptome members with distinctive qualities. RNA-seq identified thousands of circRNAs with developmental stage- and tissue-specific expression corroborating earlier suggestions that circular isoforms are a natural feature of the cell expression program. CircRNAs are abundantly expressed also in the hematopoietic compartment. There are a number of studies on circRNAs in blood cells, a specific overview is however lacking. In this review we first present current insight in circRNA biogenesis discussing the relevance for hematopoiesis of the highly interleaved processes of splicing and circRNA biogenesis. Regarding molecular functions circRNAs modulate host gene expression, but also compete for binding of microRNAs, RNA-binding proteins or translation initiation and participate in regulatory circuits. We examine circRNA expression in the hematopoietic compartment and in hematologic malignancies and review the recent breakthrough study that identified pathogenic circRNAs derived from leukemia fusion genes. CircRNA high and regulated expression in blood cell types indicate that further studies are warranted to inform the position of these regulators in normal and malignant hematopoiesis.
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Affiliation(s)
- A Bonizzato
- Department of Women's and Children's Health, University of Padova, Padova, Italy
| | - E Gaffo
- Department of Molecular Medicine, University of Padova, Padova, Italy
| | - G te Kronnie
- Department of Women's and Children's Health, University of Padova, Padova, Italy
| | - S Bortoluzzi
- Department of Molecular Medicine, University of Padova, Padova, Italy
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13
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Azizian A, Gruber J, Ghadimi BM, Gaedcke J. MicroRNA in rectal cancer. World J Gastrointest Oncol 2016; 8:416-426. [PMID: 27190581 PMCID: PMC4865709 DOI: 10.4251/wjgo.v8.i5.416] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/03/2015] [Revised: 12/01/2015] [Accepted: 03/09/2016] [Indexed: 02/05/2023] Open
Abstract
In rectal cancer, one of the most common cancers worldwide, the proper staging of the disease determines the subsequent therapy. For those with locally advanced rectal cancer, a neoadjuvant chemoradiotherapy (CRT) is recommended before any surgery. However, response to CRT ranges from complete response (responders) to complete resistance (non-responders). To date we are not able to separate in advance the first group from the second, due to the absence of a valid biomarker. Therefore all patients receive the same therapy regardless of whether they reap benefits. On the other hand almost all patients receive a surgical resection after the CRT, although a watch-and-wait procedure or an endoscopic resection might be sufficient for those who responded well to the CRT. Being highly conserved regulators of gene expression, microRNAs (miRNAs) seem to be promising candidates for biomarkers. Many studies have been analyzing the miRNAs expressed in rectal cancer tissue to determine a specific miRNA profile for the ailment. Unfortunately, there is only a small overlap of identified miRNAs between different studies, posing the question as to whether different methods or differences in tissue storage may contribute to that fact or if the results simply are not reproducible, due to unknown factors with undetected influences on miRNA expression. Other studies sought to find miRNAs which correlate to clinical parameters (tumor grade, nodal stage, metastasis, survival) and therapy response. Although several miRNAs seem to have an impact on the response to CRT or might predict nodal stage, there is still only little overlap between different studies. We here aimed to summarize the current literature on rectal cancer and miRNA expression with respect to the different relevant clinical parameters.
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14
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Butkytė S, Čiupas L, Jakubauskienė E, Vilys L, Mocevicius P, Kanopka A, Vilkaitis G. Splicing-dependent expression of microRNAs of mirtron origin in human digestive and excretory system cancer cells. Clin Epigenetics 2016; 8:33. [PMID: 27019673 PMCID: PMC4807562 DOI: 10.1186/s13148-016-0200-y] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2016] [Accepted: 03/18/2016] [Indexed: 11/17/2022] Open
Abstract
Background An abundant class of intronic microRNAs (miRNAs) undergoes atypical Drosha-independent biogenesis in which the spliceosome governs the excision of hairpin miRNA precursors, called mirtrons. Although nearly 500 splicing-dependent miRNA candidates have been recently predicted via bioinformatic analysis of human RNA-Seq datasets, only a few of them have been experimentally validated. The detailed mechanism of miRNA processing by the splicing machinery and the roles of mirtronic miRNAs in cancer are yet to be uncovered. Methods We experimentally examined whether biogenesis of certain miRNAs is under a splicing control by analyzing their expression levels in response to alterations in the 5′- and 3′-splice sites of a series of intron-containing minigenes carrying appropriate miRNAs. The expression levels of the miRNAs processed from mirtrons were determined by quantitative real-time PCR in five digestive tract (pancreas PANC-1, SU.86.86, T3M4, stomach KATOIII, colon HCT116) and two excretory system (kidney CaKi-1, 786-O) carcinoma cell lines as well as in pancreatic, stomach, and colorectal tumors. Transiently expressed SRSF1 and SRSF2 splicing factors were quantified by western blotting in the nuclear fractions of HCT116 cells. Results We found that biogenesis of the human hsa-miR-1227-3p, hsa-miR-1229-3p, and hsa-miR-1236-3p is splicing-dependent; therefore, these miRNAs can be assigned to the class of miRNAs processed by a non-canonical mirtron pathway. The expression analysis revealed a differential regulation of human mirtronic miRNAs in various cancer cell lines and tumors. In particular, hsa-miR-1229-3p is selectively upregulated in the pancreatic and stomach cancer cell lines derived from metastatic sites. Compared with the healthy controls, the expression of hsa-miR-1226-3p was significantly higher in stomach tumors but extensively downregulated in colorectal tumors. Furthermore, we provided evidence that overexpression of SRSF1 or SRSF2 can upregulate the processing of individual mirtronic miRNAs in HCT116 cells. Conclusions An interplay of different splicing factors, such as SRSF1 or SRSF2, may alter the levels of miRNAs of mirtron origin in a cell. Our findings underline the specific expression profiles of mirtronic miRNAs in colorectal, stomach, and pancreatic cancer. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0200-y) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Stasė Butkytė
- Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania
| | - Laurynas Čiupas
- Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania
| | - Eglė Jakubauskienė
- Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania
| | - Laurynas Vilys
- Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania
| | - Paulius Mocevicius
- Institute for Digestive Research, Lithuanian University of Health Sciences, Kaunas, Lithuania
| | - Arvydas Kanopka
- Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania
| | - Giedrius Vilkaitis
- Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania
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15
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Kebschull M, Papapanou PN. Mini but mighty: microRNAs in the pathobiology of periodontal disease. Periodontol 2000 2015; 69:201-20. [PMID: 26252410 PMCID: PMC4530521 DOI: 10.1111/prd.12095] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/03/2015] [Indexed: 12/19/2022]
Abstract
MicroRNAs (miRNAs) are a family of small, noncoding RNA molecules that negatively regulate protein expression either by inhibiting initiation of the translation of mRNA or by inducing the degradation of mRNA molecules. Accumulating evidence suggests that miRNA-mediated repression of protein expression is of paramount importance in a broad range of physiologic and pathologic conditions. In particular, miRNA-induced dysregulation of molecular processes involved in inflammatory pathways has been shown to contribute to the development of chronic inflammatory diseases. In this review, first of all we provide an overview of miRNA biogenesis, the main mechanisms of action and the miRNA profiling tools currently available. Then, we summarize the available evidence supporting a specific role for miRNAs in the pathobiology of periodontitis. Based on a review of available data on the differential expression of miRNAs in gingival tissues in states of periodontal health and disease, we address specific roles for miRNAs in molecular and cellular pathways causally linked to periodontitis. Our review points to several lines of evidence suggesting the involvement of miRNAs in periodontal tissue homeostasis and pathology. Although the intricate regulatory networks affected by miRNA function are still incompletely mapped, further utilization of systems biology tools is expected to enhance our understanding of the pathobiology of periodontitis.
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Affiliation(s)
- Moritz Kebschull
- Associate Professor of Dental Medicine, Consultant, Department of Periodontology, Operative and Preventive Dentistry, University of Bonn, Welschnonnenstr. 17, 53111 Bonn, Germany, Tel: +49-228-28722-007,
| | - Panos N. Papapanou
- Professor of Dental Medicine, Director, Division of Periodontics, Chair, Section of Oral and Diagnostic Sciences, Columbia University College of Dental Medicine, 630 West 168 Street, PH-7E-110, New York, NY 10032, USA, Tel: +1-212-342-3008, Fax: +1-212-305-9313,
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16
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Kock KH, Kong KW, Hoon S, Seow Y. Functional VEGFA knockdown with artificial 3'-tailed mirtrons defined by 5' splice site and branch point. Nucleic Acids Res 2015; 43:6568-78. [PMID: 26089392 PMCID: PMC4513878 DOI: 10.1093/nar/gkv617] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2015] [Accepted: 06/02/2015] [Indexed: 11/28/2022] Open
Abstract
Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs, and will be especially useful for co-delivery of coding genes and RNAi. A specific family of mirtrons – 3′-tailed mirtrons – has hairpins precisely defined on the 5′ end by the 5′ splice site and 3′ end by the branch point. Here, we present design principles for artificial 3′-tailed mirtrons and demonstrate, for the first time, efficient gene knockdown with tailed mirtrons within eGFP coding region. These artificial tailed mirtrons, unlike canonical mirtrons, have very few sequence design restrictions. Tailed mirtrons targeted against VEGFA mRNA, the misregulation of which is causative of several disorders including cancer, achieved significant levels of gene knockdown. Tailed mirtron-mediated knockdown was further shown to be splicing-dependent, and at least as effective as equivalent artificial intronic miRNAs, with the added advantage of very defined cleavage sites for generation of mature miRNA guide strands. Further development and exploitation of this unique mirtron biogenesis pathway for therapeutic RNAi coupled into protein-expressing genes can potentially enable the development of precisely controlled combinatorial gene therapy.
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Affiliation(s)
- Kian Hong Kock
- Molecular Engineering Laboratory, Biomedical Medical Sciences Institutes, 61 Biopolis Drive Proteos #03-13 Singapore 138673
| | - Kiat Whye Kong
- Molecular Engineering Laboratory, Biomedical Medical Sciences Institutes, 61 Biopolis Drive Proteos #03-13 Singapore 138673
| | - Shawn Hoon
- Molecular Engineering Laboratory, Biomedical Medical Sciences Institutes, 61 Biopolis Drive Proteos #03-13 Singapore 138673 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551
| | - Yiqi Seow
- Molecular Engineering Laboratory, Biomedical Medical Sciences Institutes, 61 Biopolis Drive Proteos #03-13 Singapore 138673
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17
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Song J, Lee JE. ASK1 modulates the expression of microRNA Let7A in microglia under high glucose in vitro condition. Front Cell Neurosci 2015; 9:198. [PMID: 26041997 PMCID: PMC4438231 DOI: 10.3389/fncel.2015.00198] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2015] [Accepted: 05/07/2015] [Indexed: 12/26/2022] Open
Abstract
Hyperglycemia results in oxidative stress and leads to neuronal apoptosis in the brain. Diabetes studies show that microglia participate in the progression of neuropathogenesis through their involvement in inflammation in vivo and in vitro. In high-glucose-induced inflammation, apoptosis signal regulating kinase 1 (ASK1) triggers the release of apoptosis cytokines and apoptotic gene expression. MicroRNA-Let7A (miR-Let7A) is reported to be a regulator of inflammation. In the present study, we investigated whether miR-Let7A regulates the function of microglia by controlling ASK1 in response to high-glucose-induced oxidative stress. We performed reverse transcription (RT) polymerase chain reaction, Taqman assay, real-time polymerase chain reaction, and immunocytochemistry to confirm the alteration of microglia function. Our results show that miR-Let7A is associated with the activation of ASK1 and the expression of anti-inflammatory cytokine (interleukin (IL)-10) and Mycs (c-Myc and N-Myc). Thus, the relationship between Let-7A and ASK1 could be a novel target for enhancing the beneficial function of microglia in central nervous system (CNS) disorders.
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Affiliation(s)
- Juhyun Song
- Department of Anatomy, Yonsei University College of Medicine Seoul, South Korea
| | - Jong Eun Lee
- Department of Anatomy, Yonsei University College of Medicine Seoul, South Korea ; Brain Korea 21 Plus Project for Medical Sciences, Brain Research Institute, Yonsei University College of Medicine Seoul, South Korea
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18
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Schamberger A, Orbán TI. Experimental validation of predicted mammalian microRNAs of mirtron origin. Methods Mol Biol 2015; 1182:245-63. [PMID: 25055917 DOI: 10.1007/978-1-4939-1062-5_22] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/11/2023]
Abstract
MicroRNAs (miRNAs) are ~22 nucleotide-long noncoding RNAs influencing many cellular processes by their regulatory functions on gene expression. MiRNAs of mirtron origin represent the most prominent group of the alternatively processed miRNAs. They reside in short introns, which are essentially equivalent to the precursor form of the given miRNA. Consequently, their maturation is independent of the Drosha/DGCR8 complex, while depends on the mechanism of mRNA splicing. The number of predicted human mirtron sequences increases as a consequence of the growing deep sequencing data and refined bioinformatics tools. However, experimental validations of particular sequences are also essential. In this chapter, we intend to provide detailed protocols for the investigation of predicted mirtron sequences. First, we use the Sleeping Beauty transposon-based gene-delivery system for the development of cell lines stably overexpressing mirtrons. The processing of functional mature miRNAs is then detected by a luciferase assay using a very strict "triple control" system. In addition, bona fide mirtron features are confirmed by demonstrating splicing dependency through splice site mutations, while Drosha/DGCR8 independency is assessed in DGCR8 deficient cell line. Finally, the presence of mirtron-derived mature miRNAs is detected by quantitative real-time PCR.
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Affiliation(s)
- Anita Schamberger
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudosok korutja 2., Budapest, 1117, Hungary
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19
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Amacher DE. Progress in the search for circulating biomarkers of nonalcoholic fatty liver disease. Biomarkers 2014; 19:541-52. [PMID: 25189636 DOI: 10.3109/1354750x.2014.958535] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
CONTEXT The definitive standard for the diagnosis of nonalcoholic fatty liver disease (NAFLD) is clinico-pathological correlation, but frequently the only laboratory abnormality is an elevation of serum aminotransferases. OBJECTIVE This has resulted in the search for more specific laboratory biomarkers. METHODS The literature was searched for novel plasma/serum markers of NAFLD. RESULTS Studies reviewed here included histologically-confirmed patients presenting some stage of NAFLD and monitored one or more novel serum/plasma biomarkers. CONCLUSION The most promising application of some of these novel biomarkers for the detection and quantification of NAFLD and particularly NASH appears to be in the combination of several into diagnostic panels.
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20
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Schamberger A, Orbán TI. 3' IsomiR species and DNA contamination influence reliable quantification of microRNAs by stem-loop quantitative PCR. PLoS One 2014; 9:e106315. [PMID: 25170848 PMCID: PMC4149544 DOI: 10.1371/journal.pone.0106315] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2013] [Accepted: 08/05/2014] [Indexed: 01/08/2023] Open
Abstract
MicroRNAs (miRNAs) are ∼20–24 nucleotide-long regulatory RNAs that have been proven to play important roles in many cellular processes. Since their discovery, a number of different techniques have been developed to detect and accurately quantify them. For individual mature miRNA measurements, quantitative stem-loop real-time PCR represents a widely used method. Although there are some data on optimization of this technique, there are still many factors that have not been investigated yet. In this study, we have thoroughly optimized this technique and pointed out several important factors that influence reliable quantification. First, we found that total RNA input can affect the measurements. Second, our data showed that carryover DNA contamination could also mislead the detection in a sequence-specific manner. Additionally, we provided evidence that different 3′ isomiR species of a particular miRNA can be reverse transcribed and cross-detected even by specifically targeted assays. Besides these, we have investigated the measurement of reaction efficiencies from total RNA samples and the accuracy of simultaneous reverse transcription reactions for increasing reliability and cost effectiveness without the loss of sensitivity and specificity. In summary, we provide a detailed, refined protocol for reliable detection of microRNA species by quantitative stem-loop PCR.
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Affiliation(s)
- Anita Schamberger
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
| | - Tamás I. Orbán
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
- Chemical Technology Transfer Ltd., Budapest, Hungary
- * E-mail:
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21
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Figueira MF, Monnerat-Cahli G, Medei E, Carvalho AB, Morales MM, Lamas ME, da Fonseca RN, Souza-Menezes J. MicroRNAs: potential therapeutic targets in diabetic complications of the cardiovascular and renal systems. Acta Physiol (Oxf) 2014; 211:491-500. [PMID: 24837225 DOI: 10.1111/apha.12316] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2014] [Revised: 02/27/2014] [Accepted: 05/12/2014] [Indexed: 12/28/2022]
Abstract
Diabetes mellitus is a serious health problem that can lead to several pathological complications in numerous organs and tissues. The most important and most prevalent organs affected by this disease are the heart and the kidneys, and these complications are the major causes of death in patients with diabetes. MicroRNAs (miRNAs), short non-coding RNAs, have been found to be functionally important in the regulation of several pathological processes, and they are emerging as an important therapeutic tool to avoid the complications of diabetes mellitus. This review summarizes the knowledge on the effects of miRNAs in diabetes. The use of miRNAs in diabetes from a clinical perspective is also discussed, focusing on their potential role to repair cardiovascular and renal complications.
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Affiliation(s)
- M. F. Figueira
- Centro de Ciências da Saúde; Instituto de Biofísica Carlos Chagas Filho; Universidade Federal do Rio de Janeiro; Rio de Janeiro Brazil
- Laboratório Integrado de Ciências Morfofuncionais; Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé; Centro de Ciências da Saúde; Universidade Federal do Rio de Janeiro; Macaé Brazil
| | - G. Monnerat-Cahli
- Centro de Ciências da Saúde; Instituto de Biofísica Carlos Chagas Filho; Universidade Federal do Rio de Janeiro; Rio de Janeiro Brazil
| | - E. Medei
- Centro de Ciências da Saúde; Instituto de Biofísica Carlos Chagas Filho; Universidade Federal do Rio de Janeiro; Rio de Janeiro Brazil
| | - A. B. Carvalho
- Centro de Ciências da Saúde; Instituto de Biofísica Carlos Chagas Filho; Universidade Federal do Rio de Janeiro; Rio de Janeiro Brazil
| | - M. M. Morales
- Centro de Ciências da Saúde; Instituto de Biofísica Carlos Chagas Filho; Universidade Federal do Rio de Janeiro; Rio de Janeiro Brazil
| | - M. E. Lamas
- Centro de Ciências da Saúde; Instituto de Biofísica Carlos Chagas Filho; Universidade Federal do Rio de Janeiro; Rio de Janeiro Brazil
| | - R. N. da Fonseca
- Laboratório Integrado de Ciências Morfofuncionais; Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé; Centro de Ciências da Saúde; Universidade Federal do Rio de Janeiro; Macaé Brazil
| | - J. Souza-Menezes
- Centro de Ciências da Saúde; Instituto de Biofísica Carlos Chagas Filho; Universidade Federal do Rio de Janeiro; Rio de Janeiro Brazil
- Laboratório Integrado de Ciências Morfofuncionais; Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé; Centro de Ciências da Saúde; Universidade Federal do Rio de Janeiro; Macaé Brazil
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