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Aye HM, Li FJ, He CY. Dynamic composition of stress granules in Trypanosoma brucei. PLoS Pathog 2024; 20:e1012666. [PMID: 39480887 PMCID: PMC11556693 DOI: 10.1371/journal.ppat.1012666] [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: 03/07/2024] [Revised: 11/12/2024] [Accepted: 10/14/2024] [Indexed: 11/02/2024] Open
Abstract
Stress granules (SGs) are stress-induced RNA condensates consisting of stalled initiation complexes resulting from translational inhibition. The biochemical composition and function of SGs are highly diverse, and this diversity has been attributed to different stress conditions, signalling pathways involved and specific cell types. Interestingly, mRNA decay components, which are found in ubiquitous cytoplasmic foci known as processing bodies (PB), have also been identified in SG proteomes. A major challenge in current SG studies is to understand the cause of SG diversity, as well as the function of SG under different stress conditions. Trypanosoma brucei is a single-cellular parasite that causes Human African Trypanosomiasis (sleeping sickness). In this study, we showed that by varying the supply of extracellular carbon sources during starvation, cellular ATP levels changed rapidly, resulting in SGs of different compositions and dynamics. We identified a subset of SG components, which dissociated from the SGs in response to cellular ATP depletion. Using expansion microscopy, we observed sub-granular compartmentalization of PB- and SG-components within the stress granules. Our results highlight the importance of cellular ATP in SG composition and dynamics, providing functional insight to SGs formed under different stress conditions.
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Affiliation(s)
- Htay Mon Aye
- Department of Biological Sciences, National University of Singapore, Singapore, Singapore
| | - Feng-Jun Li
- Department of Biological Sciences, National University of Singapore, Singapore, Singapore
| | - Cynthia Y. He
- Department of Biological Sciences, National University of Singapore, Singapore, Singapore
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2
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Bezerra MJR, Moura DMN, Freire ER, Holetz FB, Reis CRS, Monteiro TTS, Pinto ARS, Zhang N, Rezende AM, Pereira-Neves A, Figueiredo RCBQ, Clayton C, Field MC, Carrington M, de Melo Neto OP. Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei. Front Mol Biosci 2022; 9:971811. [PMID: 36275617 PMCID: PMC9585242 DOI: 10.3389/fmolb.2022.971811] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2022] [Accepted: 09/20/2022] [Indexed: 11/13/2022] Open
Abstract
Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G subunits, is responsible for the canonical selection of mRNAs required for the initiation of mRNA translation. The best-known complexes implicated in translation in trypanosomatids are based on two related pairs of eIF4E and eIF4G subunits (EIF4E3/EIF4G4 and EIF4E4/EIF4G3), whose functional distinctions remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, specifically co-precipitate with each complex. Highlights with the EIF4E4/EIF4G3 pair include RBP23, PABP1, EIF4AI and the CRK1 kinase. Co-precipitated partners with the EIF4E3/EIF4G4 pair are more diverse and include DRBD2, PABP2 and different zinc-finger proteins and RNA helicases. EIF4E3/EIF4G4 are essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes.
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Affiliation(s)
- Maria J. R. Bezerra
- Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Recife, Pernambuco, Brazil
- Department of Genetics, Federal University of Pernambuco, Recife, Pernambuco, Brazil
| | | | - Eden R. Freire
- Carlos Chagas Institute, Oswaldo Cruz Foundation, Curitiba, Pernambuco, Brazil
| | - Fabiola B. Holetz
- Carlos Chagas Institute, Oswaldo Cruz Foundation, Curitiba, Pernambuco, Brazil
| | | | | | - Adriana R. S. Pinto
- Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Recife, Pernambuco, Brazil
| | - Ning Zhang
- School of Life Sciences, University of Dundee, Dundee, United Kingdom
| | - Antonio M. Rezende
- Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Recife, Pernambuco, Brazil
| | | | | | - Christine Clayton
- Heidelberg University Center for Molecular Biology, Heidelberg, Germany
| | - Mark C. Field
- School of Life Sciences, University of Dundee, Dundee, United Kingdom
- Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czechia
| | - Mark Carrington
- Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
| | - Osvaldo P. de Melo Neto
- Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Recife, Pernambuco, Brazil
- *Correspondence: Osvaldo P. de Melo Neto,
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Inoue AH, Domingues PF, Serpeloni M, Hiraiwa PM, Vidal NM, Butterfield ER, Del Pino RC, Ludwig A, Boehm C, Field MC, Ávila AR. Proteomics Uncovers Novel Components of an Interactive Protein Network Supporting RNA Export in Trypanosomes. Mol Cell Proteomics 2022; 21:100208. [PMID: 35091090 PMCID: PMC8938319 DOI: 10.1016/j.mcpro.2022.100208] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2021] [Revised: 01/15/2022] [Accepted: 01/24/2022] [Indexed: 11/03/2022] Open
Abstract
In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation.
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Affiliation(s)
| | | | | | | | - Newton Medeiros Vidal
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA
| | | | | | - Adriana Ludwig
- Instituto Carlos Chagas, FIOCRUZ, Curitiba, Paraná, Brazil
| | - Cordula Boehm
- School of Life Sciences, University of Dundee, Dundee, Scotland, UK
| | - Mark C Field
- School of Life Sciences, University of Dundee, Dundee, Scotland, UK; Biology Centre, University of South Bohemia, České Budějovice, Czech Republic.
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4
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Abstract
In trypanosomes, RNA polymerase II transcription is polycistronic and individual mRNAs are excised by trans-splicing and polyadenylation. The lack of individual gene transcription control is compensated by control of mRNA processing, translation and degradation. Although the basic mechanisms of mRNA decay and translation are evolutionarily conserved, there are also unique aspects, such as the existence of six cap-binding translation initiation factor homologues, a novel decapping enzyme and an mRNA stabilizing complex that is recruited by RNA-binding proteins. High-throughput analyses have identified nearly a hundred regulatory mRNA-binding proteins, making trypanosomes valuable as a model system to investigate post-transcriptional regulation.
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Affiliation(s)
- Christine Clayton
- University of Heidelberg Center for Molecular Biology (ZMBH), Im Neuenheimer Feld 282, D69120 Heidelberg, Germany
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Trypanosoma brucei PRMT1 Is a Nucleic Acid Binding Protein with a Role in Energy Metabolism and the Starvation Stress Response. mBio 2018; 9:mBio.02430-18. [PMID: 30563898 PMCID: PMC6299225 DOI: 10.1128/mbio.02430-18] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
In Trypanosoma brucei and related kinetoplastid parasites, transcription of protein coding genes is largely unregulated. Rather, mRNA binding proteins, which impact processes such as transcript stability and translation efficiency, are the predominant regulators of gene expression. Arginine methylation is a posttranslational modification that preferentially targets RNA binding proteins and is, therefore, likely to have a substantial impact on T. brucei biology. The data presented here demonstrate that cells depleted of T. brucei PRMT1 (TbPRMT1), a major type I protein arginine methyltransferase, exhibit decreased virulence in an animal model. To understand the basis of this phenotype, quantitative global proteomics was employed to measure protein steady-state levels in cells lacking TbPRMT1. The approach revealed striking changes in proteins involved in energy metabolism. Most prominent were a decrease in glycolytic enzyme abundance and an increase in proline degradation pathway components, changes that resemble the metabolic remodeling that occurs during T. brucei life cycle progression. The work describes several RNA binding proteins whose association with mRNA was altered in TbPRMT1-depleted cells, and a large number of TbPRMT1-interacting proteins, thereby highlighting potential TbPRMT1 substrates. Many proteins involved in the T. brucei starvation stress response were found to interact with TbPRMT1, prompting analysis of the response of TbPRMT1-depleted cells to nutrient deprivation. Indeed, depletion of TbPRMT1 strongly hinders the ability of T. brucei to form cytoplasmic mRNA granules under starvation conditions. Finally, this work shows that TbPRMT1 itself binds nucleic acids in vitro and in vivo, a feature completely novel to protein arginine methyltransferases.IMPORTANCE Trypanosoma brucei infection causes human African trypanosomiasis, also known as sleeping sickness, a disease with a nearly 100% fatality rate when untreated. Current drugs are expensive, toxic, and highly impractical to administer, prompting the community to explore various unique aspects of T. brucei biology in search of better treatments. In this study, we identified the protein arginine methyltransferase (PRMT), TbPRMT1, as a factor that modulates numerous aspects of T. brucei biology. These include glycolysis and life cycle progression signaling, both of which are being intensely researched toward identification of potential drug targets. Our data will aid research in those fields. Furthermore, we demonstrate for the first time a direct association of a PRMT with nucleic acids, a finding we believe could translate to other organisms, including humans, thereby impacting research in fields as distant as human cancer biology and immune response modulation.
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Zeidan Q, He F, Zhang F, Zhang H, Jacobson A, Hinnebusch AG. Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo. PLoS Genet 2018; 14:e1007806. [PMID: 30532217 PMCID: PMC6307823 DOI: 10.1371/journal.pgen.1007806] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2018] [Revised: 12/27/2018] [Accepted: 11/02/2018] [Indexed: 12/21/2022] Open
Abstract
Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.
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Affiliation(s)
- Quira Zeidan
- Eunice Kennedy Shriver National Institute of Child Health and Development, National Institutes of Health, Bethesda, MD, United States of America
| | - Feng He
- Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, United States of America
| | - Fan Zhang
- Eunice Kennedy Shriver National Institute of Child Health and Development, National Institutes of Health, Bethesda, MD, United States of America
| | - Hongen Zhang
- Eunice Kennedy Shriver National Institute of Child Health and Development, National Institutes of Health, Bethesda, MD, United States of America
| | - Allan Jacobson
- Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, United States of America
| | - Alan G. Hinnebusch
- Eunice Kennedy Shriver National Institute of Child Health and Development, National Institutes of Health, Bethesda, MD, United States of America
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Romaniuk MA, Frasch AC, Cassola A. Translational repression by an RNA-binding protein promotes differentiation to infective forms in Trypanosoma cruzi. PLoS Pathog 2018; 14:e1007059. [PMID: 29864162 PMCID: PMC6002132 DOI: 10.1371/journal.ppat.1007059] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2017] [Revised: 06/14/2018] [Accepted: 04/25/2018] [Indexed: 01/08/2023] Open
Abstract
Trypanosomes, protozoan parasites of medical importance, essentially rely on post-transcriptional mechanisms to regulate gene expression in insect vectors and vertebrate hosts. RNA binding proteins (RBPs) that associate to the 3'-UTR of mature mRNAs are thought to orchestrate master developmental programs for these processes to happen. Yet, the molecular mechanisms by which differentiation occurs remain largely unexplored in these human pathogens. Here, we show that ectopic inducible expression of the RBP TcUBP1 promotes the beginning of the differentiation process from non-infective epimastigotes to infective metacyclic trypomastigotes in Trypanosoma cruzi. In early-log epimastigotes TcUBP1 promoted a drop-like phenotype, which is characterized by the presence of metacyclogenesis hallmarks, namely repositioning of the kinetoplast, the expression of an infective-stage virulence factor such as trans-sialidase, increased resistance to lysis by human complement and growth arrest. Furthermore, TcUBP1-ectopic expression in non-infective late-log epimastigotes promoted full development into metacyclic trypomastigotes. TcUBP1-derived metacyclic trypomastigotes were infective in cultured cells, and developed normally into amastigotes in the cytoplasm. By artificial in vivo tethering of TcUBP1 to the 3' untranslated region of a reporter mRNA we were able to determine that translation of the reporter was reduced by 8-fold, while its mRNA abundance was not significantly compromised. Inducible ectopic expression of TcUBP1 confirmed its role as a translational repressor, revealing significant reduction in the translation rate of multiple proteins, a reduction of polysomes, and promoting the formation of mRNA granules. Expression of TcUBP1 truncated forms revealed the requirement of both N and C-terminal glutamine-rich low complexity sequences for the development of the drop-like phenotype in early-log epimastigotes. We propose that a rise in TcUBP1 levels, in synchrony with nutritional deficiency, can promote the differentiation of T. cruzi epimastigotes into infective metacyclic trypomastigotes.
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Affiliation(s)
- Maria Albertina Romaniuk
- Instituto de Investigaciones Biotecnológicas, UNSAM-CONICET, San Martín, Provincia de Buenos Aires, Argentina
| | - Alberto Carlos Frasch
- Instituto de Investigaciones Biotecnológicas, UNSAM-CONICET, San Martín, Provincia de Buenos Aires, Argentina
| | - Alejandro Cassola
- Instituto de Investigaciones Biotecnológicas, UNSAM-CONICET, San Martín, Provincia de Buenos Aires, Argentina
- * E-mail:
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8
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Roy D, Rajyaguru PI. Suppressor of clathrin deficiency (Scd6)-An emerging RGG-motif translation repressor. WILEY INTERDISCIPLINARY REVIEWS-RNA 2018; 9:e1479. [DOI: 10.1002/wrna.1479] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/28/2017] [Revised: 03/07/2018] [Accepted: 03/07/2018] [Indexed: 12/15/2022]
Affiliation(s)
- Debadrita Roy
- Department of Biochemistry; Indian Institute of Science; Bangalore India
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9
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Abstract
5’-3’ decay is the major mRNA decay pathway in many eukaryotes, including trypanosomes. After deadenylation, mRNAs are decapped by the nudix hydrolase DCP2 of the decapping complex and finally degraded by the 5’-3’ exoribonuclease. Uniquely, trypanosomes lack homologues to all subunits of the decapping complex, while deadenylation and 5’-3’ degradation are conserved. Here, I show that the parasites use an ApaH-like phosphatase (ALPH1) as their major mRNA decapping enzyme. The protein was recently identified as a novel trypanosome stress granule protein and as involved in mRNA binding. A fraction of ALPH1 co-localises exclusively with the trypanosome 5’-3’ exoribonuclease XRNA to a special granule at the posterior pole of the cell, indicating a connection between the two enzymes. RNAi depletion of ALPH1 is lethal and causes a massive increase in total mRNAs that are deadenylated, but have not yet started 5’-3’ decay. These data suggest that ALPH1 acts downstream of deadenylation and upstream of mRNA degradation, consistent with a function in mRNA decapping. In vitro experiments show that recombinant, N-terminally truncated ALHP1 protein, but not a catalytically inactive mutant, sensitises the capped trypanosome spliced leader RNA to yeast Xrn1, but only if an RNA 5’ polyphosphatase is included. This indicates that the decapping mechanism of ALPH1 differs from the decapping mechanism of Dcp2 by leaving more than one phosphate group at the mRNA’s 5’ end. This is the first reported function of a eukaryotic ApaH-like phosphatase, a bacterial-derived class of enzymes present in all phylogenetic super-groups of the eukaryotic kingdom. The substrates of eukaryotic ApaH-like phosphatases are unknown. However, the substrate of the related bacterial enzyme ApaH, diadenosine tetraphosphate, is highly reminiscent of a eukaryotic mRNA cap. Eukaryotic mRNAs are stabilised by a 5’ cap and one important step in mRNA decay is the removal of this cap by the nudix domain protein Dcp2 of the decapping complex. The decapping complex is highly conserved throughout eukaryotes, with the exception of trypanosomes that lack the entire complex. Here, I show that trypanosomes have evolved to use an ApaH-like phosphatase instead of a nudix domain protein as their major decapping enzyme. This work closes an important gap in the knowledge of trypanosome mRNA metabolism. Moreover, this is the first reported function of an ApaH-like phosphatase, a bacterial derived class of enzymes that are widespread throughout eukaryotes.
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Affiliation(s)
- Susanne Kramer
- Biocenter, University of Würzburg, Am Hubland, Würzburg, Germany
- * E-mail:
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Romaniuk MA, Cervini G, Cassola A. Regulation of RNA binding proteins in trypanosomatid protozoan parasites. World J Biol Chem 2016; 7:146-157. [PMID: 26981203 PMCID: PMC4768119 DOI: 10.4331/wjbc.v7.i1.146] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Revised: 08/04/2015] [Accepted: 01/29/2016] [Indexed: 02/05/2023] Open
Abstract
Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3’ untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.
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Cassola A, Romaniuk MA, Primrose D, Cervini G, D'Orso I, Frasch AC. Association of UBP1 to ribonucleoprotein complexes is regulated by interaction with the trypanosome ortholog of the human multifunctional P32 protein. Mol Microbiol 2015; 97:1079-96. [PMID: 26096620 DOI: 10.1111/mmi.13090] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/09/2015] [Indexed: 12/30/2022]
Abstract
Regulation of gene expression in trypanosomatid parasitic protozoa is mainly achieved posttranscriptionally. RNA-binding proteins (RBPs) associate to 3' untranslated regions in mRNAs through dedicated domains such as the RNA recognition motif (RRM). Trypanosoma cruzi UBP1 (TcUBP1) is an RRM-type RBP involved in stabilization/degradation of mRNAs. TcUBP1 uses its RRM to associate with cytoplasmic mRNA and to mRNA granules under starvation stress. Here, we show that under starvation stress, TcUBP1 is tightly associated with condensed cytoplasmic mRNA granules. Conversely, under high nutrient/low density-growing conditions, TcUBP1 ribonucleoprotein (RNP) complexes are lax and permeable to mRNA degradation and disassembly. After dissociating from mRNA, TcUBP1 can be phosphorylated only in unstressed parasites. We have identified TcP22, the ortholog of mammalian P32/C1QBP, as an interactor of TcUBP1 RRM. Overexpression of TcP22 decreased the number of TcUBP1 granules in starved parasites in vivo. Endogenous TcUBP1 RNP complexes could be dissociated in vitro by addition of recombinant TcP22, a condition stimulating TcUBP1 phosphorylation. Biochemical and in silico analysis revealed that TcP22 interacts with the RNA-binding surface of TcUBP1 RRM. We propose a model for the decondensation of TcUBP1 RNP complexes in T. cruzi through direct interaction with TcP22 and phosphorylation.
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Affiliation(s)
- Alejandro Cassola
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, UNSAM-CONICET, Buenos Aires, Argentina
| | - María Albertina Romaniuk
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, UNSAM-CONICET, Buenos Aires, Argentina
| | - Debora Primrose
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, UNSAM-CONICET, Buenos Aires, Argentina
| | - Gabriela Cervini
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, UNSAM-CONICET, Buenos Aires, Argentina
| | - Iván D'Orso
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, UNSAM-CONICET, Buenos Aires, Argentina
| | - Alberto Carlos Frasch
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, UNSAM-CONICET, Buenos Aires, Argentina
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12
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Guerreiro A, Deligianni E, Santos JM, Silva PAGC, Louis C, Pain A, Janse CJ, Franke-Fayard B, Carret CK, Siden-Kiamos I, Mair GR. Genome-wide RIP-Chip analysis of translational repressor-bound mRNAs in the Plasmodium gametocyte. Genome Biol 2014; 15:493. [PMID: 25418785 PMCID: PMC4234863 DOI: 10.1186/s13059-014-0493-0] [Citation(s) in RCA: 71] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2014] [Accepted: 10/09/2014] [Indexed: 01/20/2023] Open
Abstract
BACKGROUND Following fertilization, the early proteomes of metazoans are defined by the translation of stored but repressed transcripts; further embryonic development relies on de novo transcription of the zygotic genome. During sexual development of Plasmodium berghei, a rodent model for human malaria species including P. falciparum, the stability of repressed mRNAs requires the translational repressors DOZI and CITH. When these repressors are absent, Plasmodium zygote development and transmission to the mosquito vector is halted, as hundreds of transcripts become destabilized. However, which mRNAs are direct targets of these RNA binding proteins, and thus subject to translational repression, is unknown. RESULTS We identify the maternal mRNA contribution to post-fertilization development of P. berghei using RNA immunoprecipitation and microarray analysis. We find that 731 mRNAs, approximately 50% of the transcriptome, are associated with DOZI and CITH, allowing zygote development to proceed in the absence of RNA polymerase II transcription. Using GFP-tagging, we validate the repression phenotype of selected genes and identify mRNAs relying on the 5' untranslated region for translational control. Gene deletion reveals a novel protein located in the ookinete crystalloid with an essential function for sporozoite development. CONCLUSIONS Our study details for the first time the P. berghei maternal repressome. This mRNA population provides the developing ookinete with coding potential for key molecules required for life-cycle progression, and that are likely to be critical for the transmission of the malaria parasite from the rodent and the human host to the mosquito vector.
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Affiliation(s)
- Ana Guerreiro
- />Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal
| | - Elena Deligianni
- />Institute of Molecular Biology and Biotechnology (IMBB), Foundation of Research and Technology (FORTH), N. Plastira 100, Heraklio, Crete P.C. 71110 Greece
| | - Jorge M Santos
- />Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal
| | - Patricia AGC Silva
- />Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal
| | - Christos Louis
- />Institute of Molecular Biology and Biotechnology (IMBB), Foundation of Research and Technology (FORTH), N. Plastira 100, Heraklio, Crete P.C. 71110 Greece
| | - Arnab Pain
- />Pathogen Genomics Laboratory, Computational Bioscience Research Center, King Abdullah University of Science and Technology, Thuwal-Jeddah, Saudi Arabia
| | - Chris J Janse
- />Department of Parasitology, Leiden University Medical Centre, Leiden, The Netherlands
| | | | - Celine K Carret
- />Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal
| | - Inga Siden-Kiamos
- />Institute of Molecular Biology and Biotechnology (IMBB), Foundation of Research and Technology (FORTH), N. Plastira 100, Heraklio, Crete P.C. 71110 Greece
| | - Gunnar R Mair
- />Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal
- />Parasitology, Department of Infectious Diseases, University of Heidelberg Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
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Lott K, Zhu L, Fisk JC, Tomasello DL, Read LK. Functional interplay between protein arginine methyltransferases in Trypanosoma brucei. Microbiologyopen 2014; 3:595-609. [PMID: 25044453 PMCID: PMC4234254 DOI: 10.1002/mbo3.191] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2014] [Revised: 05/05/2014] [Accepted: 05/23/2014] [Indexed: 12/21/2022] Open
Abstract
Arginine methylation is a common posttranslational modification that has far-reaching cellular effects. Trypanosoma brucei is an early-branching eukaryote with four characterized protein arginine methyltransferases (PRMTs), one additional putative PRMT, and over 800 arginine methylated proteins, suggesting that arginine methylation has widespread impacts in this organism. While much is known about the activities of individual T. brucei PRMTs (TbPRMTs), little is known regarding how TbPRMTs function together in vivo. In this study, we analyzed single and selected double TbPRMT knockdowns for the impact on expression of TbPRMTs and global methylation status. Repression of TbPRMT1 caused a decrease in asymmetric dimethylarginine and a marked increase in monomethylarginine that was catalyzed by TbPRMT7, suggesting that TbPRMT1 and TbPRMT7 can compete for the same substrate. We also observed an unexpected and strong interdependence between TbPRMT1 and TbPRMT3 protein levels. This finding, together with the observation of similar methyl landscape profiles in TbPRMT1 and TbPRMT3 repressed cells, strongly suggests that these two enzymes form a functional complex. We show that corepression of TbPRMT6/7 synergistically impacts growth of procyclic-form T. brucei. Our findings also implicate the actions of noncanonical, and as yet unidentified, PRMTs in T. brucei. Together, our studies indicate that TbPRMTs display a functional interplay at multiple levels.
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Affiliation(s)
- Kaylen Lott
- Department of Microbiology and Immunology, University at Buffalo School of Medicine, Buffalo, New York, 14214
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Clayton CE. Networks of gene expression regulation in Trypanosoma brucei. Mol Biochem Parasitol 2014; 195:96-106. [PMID: 24995711 DOI: 10.1016/j.molbiopara.2014.06.005] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2014] [Revised: 06/19/2014] [Accepted: 06/23/2014] [Indexed: 10/25/2022]
Abstract
Regulation of gene expression in Kinetoplastids relies mainly on post-transcriptional mechanisms. Recent high-throughput analyses, combined with mathematical modelling, have demonstrated possibilities for transcript-specific regulation at every stage: trans splicing, polyadenylation, translation, and degradation of both the precursor and the mature mRNA. Different mRNA degradation pathways result in different types of degradation kinetics. The original idea that the fate of an mRNA - or even just its degradation kinetics - can be defined by a single "regulatory element" is an over-simplification. It is now clear that every mRNA can bind many different proteins, some of which may compete with each other. Superimposed upon this complexity are the interactions of those proteins with effectors of gene expression. The amount of protein that is made from a gene is therefore determined by a complex network of interactions.
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Affiliation(s)
- C E Clayton
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
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