A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei

Abstract

In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5′-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of “halfmer” disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames. In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5′-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of “halfmer” disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames. Trypanosoma brucei is a unicellular eukaryote that proliferates in the blood and tissue fluids of mammals and in the digestive system of tsetse flies. Trypanosoma brucei and related trypanosomes cause African sleeping sickness in humans and nagana in cattle, diseases which produce a significant economic burden for a vast region of Africa. Two T. brucei life-cycle stages are easily cultured in the laboratory. Bloodstream forms (BF) can be grown in high-glucose medium at 37°C, whereas procyclic forms are cultivated in high-proline medium at 27°C. Kinetoplastids rely almost exclusively on posttranscriptional mechanisms for control of gene expression. Transcription is polycistronic and individual mRNAs are generated by trans splicing of a 39-nucleotide leader to the 5′-end and by 3′ polyadenylation (1Clayton C. Regulation of gene expression in trypanosomatids: Living with polycistronic transcription.Open Biol. 2019; 9 (31164043): 19007210.1098/rsob.190072Crossref PubMed Scopus (45) Google Scholar). Trypanosome mRNAs vary extensively in decay rates and translation efficiency. Work by numerous laboratories has demonstrated that RNA-binding proteins (RBPs) play prominent roles in the regulation of splicing, translation, and mRNA decay, although the mechanisms by which they do this have, with a few exceptions, remained obscure (1Clayton C. Regulation of gene expression in trypanosomatids: Living with polycistronic transcription.Open Biol. 2019; 9 (31164043): 19007210.1098/rsob.190072Crossref PubMed Scopus (45) Google Scholar). Two high-throughput approaches enabled us to identify RBPs that can increase or decrease expression of bound mRNAs. The tethering assay involves co-expression of the protein of interest fused to a highly sequence-specific RNA-binding peptide and a reporter mRNA bearing the cognate recognition sequence; we used the λN peptide–boxB combination. We conducted genome-wide library screens to reveal protein fragments that can either activate or repress expression of a reporter when tethered (2Erben E.D. Fadda A. Lueong S. Hoheisel J.D. Clayton C. A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei.PLoS Pathog. 2014; 10 (24945722): e100417810.1371/journal.ppat.1004178Crossref PubMed Scopus (70) Google Scholar), results that were later verified for a subset of full-length proteins (3Lueong S. Merce C. Fischer B. Hoheisel J.D. Erben E.D. Gene expression regulatory networks in Trypanosoma brucei: Insights into the role of the mRNA-binding proteome.Mol. Microbiol. 2016; 100 (26784394): 457-47110.1111/mmi.13328Crossref PubMed Scopus (67) Google Scholar). In addition, a catalog of proteins that associate with BF mRNAs (the trypanosome mRNP proteome) was obtained (3Lueong S. Merce C. Fischer B. Hoheisel J.D. Erben E.D. Gene expression regulatory networks in Trypanosoma brucei: Insights into the role of the mRNA-binding proteome.Mol. Microbiol. 2016; 100 (26784394): 457-47110.1111/mmi.13328Crossref PubMed Scopus (67) Google Scholar). In this method, cells are irradiated with UV light to crosslink proteins to nucleic acids, then polyadenylated (poly(A)+) RNAs are purified via hybridization to oligo(dT) beads under denaturing conditions. The proteins directly bound to poly(A)+ RNAs are then identified and quantified by MS (4Castello A. Horos R. Strein C. Fischer B. Eichelbaum K. Steinmetz L.M. Krijgsveld J. Hentze M.W. System-wide identification of RNA-binding proteins by interactome capture.Nat. PubMed Scopus Google Scholar). to the identification of RBPs and and potential novel RNA-binding of the identified RBPs are essential for gene expression expression or in C. Regulation of gene expression in trypanosomatids: Living with polycistronic transcription.Open Biol. 2019; 9 (31164043): 19007210.1098/rsob.190072Crossref PubMed Scopus (45) Google Scholar), only a of a on expression when tethered to the mRNA that an when tethered is a cytoplasmic protein ZC3H5 (2Erben E.D. Fadda A. Lueong S. Hoheisel J.D. Clayton C. A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei.PLoS Pathog. 2014; 10 (24945722): e100417810.1371/journal.ppat.1004178Crossref PubMed Scopus (70) Google S. Merce C. Fischer B. Hoheisel J.D. Erben E.D. Gene expression regulatory networks in Trypanosoma brucei: Insights into the role of the mRNA-binding proteome.Mol. Microbiol. 2016; 100 (26784394): 457-47110.1111/mmi.13328Crossref PubMed Scopus (67) Google S. J.D. A trypanosome genome-wide protein PubMed Scopus Google Scholar). ZC3H5 can be and poly(A)+ RNA (3Lueong S. Merce C. Fischer B. Hoheisel J.D. Erben E.D. Gene expression regulatory networks in Trypanosoma brucei: Insights into the role of the mRNA-binding proteome.Mol. Microbiol. 2016; 100 (26784394): 457-47110.1111/mmi.13328Crossref PubMed Scopus (67) Google decreases gene expression when tethered to a reporter (3Lueong S. Merce C. Fischer B. Hoheisel J.D. Erben E.D. Gene expression regulatory networks in Trypanosoma brucei: Insights into the role of the mRNA-binding proteome.Mol. Microbiol. 2016; 100 (26784394): 457-47110.1111/mmi.13328Crossref PubMed Scopus (67) Google Scholar). expression is in BF and procyclic and high-throughput RNAi analysis that is essential in BF S. A. C. of RNA in the African PubMed Scopus Google Scholar). In addition, a yeast two-hybrid screen that ZC3H5 with the with the in the tethering assay (3Lueong S. Merce C. Fischer B. Hoheisel J.D. Erben E.D. Gene expression regulatory networks in Trypanosoma brucei: Insights into the role of the mRNA-binding proteome.Mol. Microbiol. 2016; 100 (26784394): 457-47110.1111/mmi.13328Crossref PubMed Scopus (67) Google Scholar). In this we the role of ZC3H5 in gene expression in T. We found that ZC3H5 is of a protein complex which is by depletion of ZC3H5 or other of the complex results in cells to ZC3H5 preferentially with poorly translated, low-stability mRNAs, its depletion results in only minor to the We that ZC3H5 is involved in quality control during the translation of suboptimal ZC3H5 is a protein with a ZC3H5 is the but in of and Trypanosoma reveals that the the are and is and are The whereas the are ZC3H5 has an We verified the cytoplasmic of a cell with in protein We that ZC3H5 is for BF trypanosome we a purification and in one ZC3H5 of RNAi caused a in expression and cell The of ZC3H5 was by and and The and was with and was used to the cell for cells were for cell cells with a and are in or cells with two and one are in and cells with two and two are or revealed that the cells were of of RNAi cells were to and The of cells RNAi the of cells this that the cells ZC3H5 However, the of cells in RNAi this was followed by cell death. ZC3H5 we purified ZC3H5 and the to proteins that were to we the results with a purification of K. K. Clayton Erben The mRNA-binding proteins and in Trypanosoma PubMed Scopus Google Scholar), a RNA-binding protein that to protein mRNAs. The results revealed of ZC3H5 with the proteins encoded by Tb927.11.4900, and and of the three found proteins with MS analysis that they a complex ZC3H5 was not that only a of is with of in of Tb927.11.4900, Tb927.8.1500, and in of ZC3H5 although remained in the The results that a of ZC3H5 the three proteins were in the mRNP (3Lueong S. Merce C. Fischer B. Hoheisel J.D. Erben E.D. Gene expression regulatory networks in Trypanosoma brucei: Insights into the role of the mRNA-binding proteome.Mol. Microbiol. 2016; 100 (26784394): 457-47110.1111/mmi.13328Crossref PubMed Scopus (67) Google and and are cytoplasmic when fused to S. J.D. A trypanosome genome-wide protein PubMed Scopus Google Scholar). MS ZC3H5 with the RNA-binding protein we not that in We the of the identified proteins by the in of as as the related J. T. J. J. of in 2014; PubMed Scopus Google Scholar). is a protein which has was to in procyclic forms J. S. C. A. S. into by the as a PubMed Scopus Google and is as as B. The to the which proteins implicated in The protein is The reveals three regions at the and at and brucei The regions are very and and is in but in is a protein and the as a of the and but an and the region of of the this and and is to that in and which the motif B. B. of in for RNA PubMed Scopus Google A. of the a PubMed Scopus Google Scholar). 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Fadda A. Lueong S. Hoheisel J.D. Clayton C. A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei.PLoS Pathog. 2014; 10 (24945722): e100417810.1371/journal.ppat.1004178Crossref PubMed Scopus (70) Google Scholar), we the individual full-length do trypanosomes that an mRNA the reporter with in its were with λN As a we used protein was one of the of gene expression in tethering screens (2Erben E.D. Fadda A. Lueong S. Hoheisel J.D. Clayton C. A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei.PLoS Pathog. 2014; 10 (24945722): e100417810.1371/journal.ppat.1004178Crossref PubMed Scopus (70) Google S. Merce C. Fischer B. Hoheisel J.D. Erben E.D. Gene expression regulatory networks in Trypanosoma brucei: Insights into the role of the mRNA-binding proteome.Mol. Microbiol. 2016; 100 (26784394): 457-47110.1111/mmi.13328Crossref PubMed Scopus (67) Google Scholar), caused by its with translation B. Clayton C. The and in Trypanosoma Biol. 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J. for the of RNAi in Trypanosoma PubMed Scopus Google Scholar). were with a for into the of Trypanosoma brucei cell and identified a RNAi Pathog. 2014; 10 PubMed Scopus Google Scholar). The was with and and the containing the was into the the tethering cell reporter with were with Tb927.8.1500, and in with the (2Erben E.D. Fadda A. Lueong S. Hoheisel J.D. Clayton C. A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei.PLoS Pathog. 2014; 10 (24945722): e100417810.1371/journal.ppat.1004178Crossref PubMed Scopus (70) Google S. Merce C. Fischer B. Hoheisel J.D. Erben E.D. Gene expression regulatory networks in Trypanosoma brucei: Insights into the role of the mRNA-binding proteome.Mol. Microbiol. 2016; 100 (26784394): 457-47110.1111/mmi.13328Crossref PubMed Scopus (67) Google Scholar). were in the of at the and 10 and obtained by yeast were into the R. C. J. K. of a reading yeast two-hybrid system for and protein PubMed Scopus Google and pairwise analysis as previously (3Lueong S. Merce C. Fischer B. Hoheisel J.D. Erben E.D. Gene expression regulatory networks in Trypanosoma brucei: Insights into the role of the mRNA-binding proteome.Mol. Microbiol. 2016; 100 (26784394): 457-47110.1111/mmi.13328Crossref PubMed Scopus (67) Google Scholar). The and were generated the the of and are found in were by and by to the were the the and was in a assay of the to the of Erben E.D. The tethering A for the of Biol. PubMed Scopus Google Scholar). of K. K. Clayton Erben The mRNA-binding proteins and in Trypanosoma PubMed Scopus Google Scholar), Tb927.8.1500, and proteins were as previously A. Fadda A. Clayton C. Erben Trypanosome and the RNA-binding protein and potential roles in post-transcriptional regulatory 2014; PubMed Scopus Google Scholar). purification of in ZC3H5 was to and Clayton Clayton C. of the RNA-binding protein the in Trypanosoma brucei.PLoS Pathog. PubMed Scopus Google Scholar). proteins were on to and by proteins were in three by by the via the system directly to an were to an with The was in MS and were J. peptide identification and protein PubMed Scopus Google and for and for peptide identification and were the T. brucei protein and for and of were We of as and of of and as and as the with to two The for proteins and was a of two protein and a peptide of were and were to the protein was in individual for with the of the to and for the and other are of analysis of the results was the S. T. A. T. J. The for analysis of 2016; PubMed Scopus Google Scholar). were to a of two of three in one this were by and were used the of the the and the control K. K. 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