Advertisement

Basal Splicing Factors Regulate the Stability of Mature mRNAs in Trypanosomes*

  • Sachin Kumar Gupta
    Affiliations
    From the Mina and Everard Goodman Faculty of Life Sciences, and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
    Search for articles by this author
  • Shai Carmi
    Footnotes
    Affiliations
    From the Mina and Everard Goodman Faculty of Life Sciences, and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
    Search for articles by this author
  • Hiba Waldman Ben-Asher
    Affiliations
    From the Mina and Everard Goodman Faculty of Life Sciences, and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
    Search for articles by this author
  • Itai Dov Tkacz
    Affiliations
    From the Mina and Everard Goodman Faculty of Life Sciences, and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
    Search for articles by this author
  • Ilana Naboishchikov
    Affiliations
    From the Mina and Everard Goodman Faculty of Life Sciences, and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
    Search for articles by this author
  • Shulamit Michaeli
    Correspondence
    Holds the David and Inez Myers Chair in RNA Silencing of Diseases. To whom correspondence should be addressed. Tel.: 972-3-5318068; Fax: 972-3-7384058;
    Affiliations
    From the Mina and Everard Goodman Faculty of Life Sciences, and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by the Deutsche Forcshungsgemeinschaft via Deutsche-Israelische Projektkooperation and the Israel Science Foundation.
    This article contains supplemental S-1–S-4 and additional references.
    1 Supported by the Human Frontier Science Program.
Open AccessPublished:January 02, 2013DOI:https://doi.org/10.1074/jbc.M112.416578
      Gene expression in trypanosomes is mainly regulated post-transcriptionally. Genes are transcribed as polycistronic mRNAs that are dissected by the concerted action of trans-splicing and polyadenylation. In trans-splicing, a common exon, the spliced leader, is added to all mRNAs from a small RNA. In this study, we examined by microarray analysis the transcriptome following RNAi silencing of the basal splicing factors U2AF65, SF1, and U2AF35. The transcriptome data revealed correlations between the affected genes and their splicing and polyadenylation signaling properties, suggesting that differential binding of these factors to pre-mRNA regulates trans-splicing and hence expression of specific genes. Surprisingly, all these factors were shown to affect not only splicing but also mRNA stability. Affinity purification of SF1 and U2AF35 complexes supported their role in mRNA stability. U2AF35 but not SF1 was shown to bind to ribosomes. To examine the role of splicing factors in mRNA stability, mutations were introduced into the polypyrimidine tract located in the 3′ UTR of a mini-gene, and the results demonstrate that U2AF65 binds to such a site and controls the mRNA stability. We propose that transcripts carrying splicing signals in their 3′ UTR bind the splicing factors and control their stability.
      Background: Trypanosome trans-splicing depends on basal splicing factors such as U2AF35, U2FA65, and SF1.
      Results: Transcriptome analyses of RNAi-silenced cells of basal splicing factors reveal differential reliance on factors for trans-splicing and a role for the splicing factors in mRNA stability.
      Conclusion: Basal splicing factors regulate trans-splicing and mRNA stability.
      Significance: This is the first study to suggest that basal splicing factors regulate mRNA stability.

      Introduction

      Trypanosomes are unicellular parasites that cause devastating diseases. Trypanosoma brucei is the causative agent of sleeping sickness in humans. The parasite cycles between the insect host (procyclic form) and the mammalian host (bloodstream form). mRNA processing in trypanosomes differs from this process in other eukaryotes. All mRNAs are trans-spliced, although two cis-introns have been identified. No distinct promoters for protein-coding genes exist, and thus, all mRNAs are transcribed as long polycistronic units. trans-Splicing, in conjunction with polyadenylation, functions to dissect the polycistronic transcripts into individual mRNAs. In trans-splicing, a small exon, the spliced leader (SL),
      The abbreviations used are: SL
      spliced leader
      PPT
      polypyrimidine tract
      RNP
      ribonucleoprotein
      hnRNP
      heterogeneous nuclear RNP
      PTB
      polypyrimidine tract binding
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      nt
      nucleotide
      RRM
      RNA recognition motif
      TAP
      tandem affinity purification.
      is added to all mRNAs from a small RNA, the SL RNA (
      • Agabian N.
      Trans-splicing of nuclear pre-mRNAs.
      ,
      • Liang X.H.
      • Haritan A.
      • Uliel S.
      • Michaeli S.
      Trans- and cis-splicing in trypanosomatids. Mechanism, factors, and regulation.
      ,
      • Michaeli S.
      Trans-splicing in trypanosomes. Machinery and its impact on the parasite transcriptome.
      ).
      Several recent studies shed light on the contribution of trans-splicing and polyadenylation to global gene expression in these parasites. Together, these studies identified alternative processing of transcripts at either their 5′ end or, in a larger number of cases, at their 3′ end (
      • Kolev N.G.
      • Franklin J.B.
      • Carmi S.
      • Shi H.
      • Michaeli S.
      • Tschudi C.
      The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.
      ,
      • Siegel T.N.
      • Hekstra D.R.
      • Wang X.
      • Dewell S.
      • Cross G.A.
      Genome-wide analysis of mRNA abundance in two life-cycle stages of Trypanosoma brucei and identification of splicing and polyadenylation sites.
      ). The sequences that determine the choice of splice site are the AG splice site and the polypyrimidine tract (PPT). Heterogeneity in the composition of the PPT and its distance from the AG splice site might regulate trans-splicing events (
      • Kolev N.G.
      • Franklin J.B.
      • Carmi S.
      • Shi H.
      • Michaeli S.
      • Tschudi C.
      The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.
      ,
      • Siegel T.N.
      • Hekstra D.R.
      • Wang X.
      • Dewell S.
      • Cross G.A.
      Genome-wide analysis of mRNA abundance in two life-cycle stages of Trypanosoma brucei and identification of splicing and polyadenylation sites.
      ,
      • Nilsson D.
      • Gunasekera K.
      • Mani J.
      • Osteras M.
      • Farinelli L.
      • Baerlocher L.
      • Roditi I.
      • Ochsenreiter T.
      Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei.
      ). Despite these recent studies, the most robust mechanism that was shown so far to regulate the trypanosome transcriptome is mRNA stability (
      • Kramer S.
      • Carrington M.
      Trans-acting proteins regulating mRNA maturation, stability, and translation in trypanosomatids.
      ,
      • Clayton C.
      • Shapira M.
      Post-transcriptional regulation of gene expression in trypanosomes and leishmanias.
      ).
      Relatively little is known regarding the contribution of splicing factors to regulating trans-splicing in trypanosomes. The most highly conserved basal factors that bind to the pre-mRNA are essential for both cis- and trans-splicing (
      • Liang X.H.
      • Haritan A.
      • Uliel S.
      • Michaeli S.
      Trans- and cis-splicing in trypanosomatids. Mechanism, factors, and regulation.
      ,
      • Michaeli S.
      Trans-splicing in trypanosomes. Machinery and its impact on the parasite transcriptome.
      ,
      • Günzl A.
      The pre-mRNA splicing machinery of trypanosomes. Complex or simplified?.
      ). The trypanosome U2AF65, which binds to PPT, differs from its homologues in metazoa. The protein contains three RNA recognition motif (RRM) domains, but the RRMIII includes an insertion of a hydrophobic region lacking secondary structure that was classified as a nonregular secondary structure. This nonregular secondary structure sequence was shown to be essential for the interaction of U2AF65 and SF1 in a yeast two-hybrid assay, and the proteins were shown to interact in vivo (
      • Vazquez M.P.
      • Mualem D.
      • Bercovich N.
      • Stern M.Z.
      • Nyambega B.
      • Barda O.
      • Nasiga D.
      • Gupta S.K.
      • Michaeli S.
      • Levin M.J.
      Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65, and SF1.
      ). The trypanosome U2AF35, which is believed to interact with the 3′ splice site (AG), lacks the residues that mediate the interaction with U2AF65 to form a heterodimeric complex like in metazoa (
      • Vázquez M.
      • Atorrasagasti C.
      • Bercovich N.
      • Volcovich R.
      • Levin M.J.
      Unique features of the Trypanosoma cruzi U2AF35 splicing factor.
      ). Tryptophan 134 needed for this reciprocal “tongue in groove” heterodimerization is mutated to lysine in the trypanosome U2AF35 (
      • Vázquez M.
      • Atorrasagasti C.
      • Bercovich N.
      • Volcovich R.
      • Levin M.J.
      Unique features of the Trypanosoma cruzi U2AF35 splicing factor.
      ), and thus, these trypanosome proteins do not interact in vivo (
      • Vazquez M.P.
      • Mualem D.
      • Bercovich N.
      • Stern M.Z.
      • Nyambega B.
      • Barda O.
      • Nasiga D.
      • Gupta S.K.
      • Michaeli S.
      • Levin M.J.
      Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65, and SF1.
      ). Silencing of U2AF35, U2AF65, and SF1 compromises trans- and cis-splicing, but interestingly, silencing of U2AF35 and SF1 leads to accumulation of SL RNA, whereas SL RNA level is reduced in U2AF65-silenced cells. U2AF65 may therefore have a very special role in trans-splicing and may participate in the recruitment of SL RNP to the spliceosome (
      • Michaeli S.
      Trans-splicing in trypanosomes. Machinery and its impact on the parasite transcriptome.
      ). The obligatory interaction between SF1-U2AF35-U2AF65 in mammals forces a very strict spatial distance between the 3′ splice site, PPT, and the branch site (
      • Kol G.
      • Lev-Maor G.
      • Ast G.
      Human-mouse comparative analysis reveals that branch-site plasticity contributes to splicing regulation.
      ,
      • Schwartz S.H.
      • Silva J.
      • Burstein D.
      • Pupko T.
      • Eyras E.
      • Ast G.
      Large scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes.
      ). However, in trypanosomes, this is not the case, and flexibility exists in the distance between the 3′ splice site and the PPT tract (
      • Kolev N.G.
      • Franklin J.B.
      • Carmi S.
      • Shi H.
      • Michaeli S.
      • Tschudi C.
      The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.
      ,
      • Siegel T.N.
      • Hekstra D.R.
      • Wang X.
      • Dewell S.
      • Cross G.A.
      Genome-wide analysis of mRNA abundance in two life-cycle stages of Trypanosoma brucei and identification of splicing and polyadenylation sites.
      ). This spatial flexibility may provide room for binding of protein(s) that regulate trans-splicing.
      Several RNA-binding proteins that were shown to participate in splicing regulation in metazoa also exist in trypanosomes. Among these are heterogeneous nuclear ribonucleoprotein (hnRNP) and proteins carrying a serine-arginine motif (SR) (
      • Liang X.H.
      • Haritan A.
      • Uliel S.
      • Michaeli S.
      Trans- and cis-splicing in trypanosomatids. Mechanism, factors, and regulation.
      ,
      • Michaeli S.
      Trans-splicing in trypanosomes. Machinery and its impact on the parasite transcriptome.
      ,
      • Günzl A.
      The pre-mRNA splicing machinery of trypanosomes. Complex or simplified?.
      ). Three SR proteins were described in trypanosomes as follows: TSR1, TSR1IP, and TRRM1 (
      • Ismaïli N.
      • Pérez-Morga D.
      • Walsh P.
      • Cadogan M.
      • Pays A.
      • Tebabi P.
      • Pays E.
      Characterization of a Trypanosoma brucei SR domain-containing protein bearing homology to cis-spliceosomal U1 70-kDa proteins.
      ,
      • Ismaïli N.
      • Pérez-Morga D.
      • Walsh P.
      • Mayeda A.
      • Pays A.
      • Tebabi P.
      • Krainer A.R.
      • Pays E.
      Characterization of a SR protein from Trypanosoma brucei with homology to RNA-binding cis-splicing proteins.
      ,
      • Manger I.D.
      • Boothroyd J.C.
      Identification of a nuclear protein in Trypanosoma brucei with homology to RNA-binding proteins from cis-splicing systems.
      ). However, their exact role in trans-splicing is not currently known. Polypyrimidine tract-binding proteins (PTB) or hnRNPI homologues were shown to be required for trans-splicing of mRNAs carrying a C-rich polypyrimidine (
      • Estévez A.M.
      The RNA-binding protein TbDRBD3 regulates the stability of a specific subset of mRNAs in trypanosomes.
      ,
      • Stern M.Z.
      • Gupta S.K.
      • Salmon-Divon M.
      • Haham T.
      • Barda O.
      • Levi S.
      • Wachtel C.
      • Nilsen T.W.
      • Michaeli S.
      Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism.
      ). The PTB proteins were also shown to regulate mRNA stability (
      • Estévez A.M.
      The RNA-binding protein TbDRBD3 regulates the stability of a specific subset of mRNAs in trypanosomes.
      ,
      • Stern M.Z.
      • Gupta S.K.
      • Salmon-Divon M.
      • Haham T.
      • Barda O.
      • Levi S.
      • Wachtel C.
      • Nilsen T.W.
      • Michaeli S.
      Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism.
      ).
      In this study, the role of the T. brucei basal splicing factors U2AF35, U2AF65, and SF1 on the transcriptome was examined in cells depleted of these factors by RNAi. The basal splicing factors were shown to differentially affect the transcriptome, suggesting that each of these factors controls different groups of genes. Bioinformatic studies were conducted to identify a correlation between the affected genes and the properties of their SL and poly(A) addition sites and spatial organization of their splicing signals. Indeed, U2AF65 and SF1 target genes were shown to undergo differential polyadenylation and to possess a short PPT poor in pyrimidines. Over a third of the transcripts previously shown to undergo differential polyadenylation (
      • Kolev N.G.
      • Franklin J.B.
      • Carmi S.
      • Shi H.
      • Michaeli S.
      • Tschudi C.
      The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.
      ) were regulated by the splicing factors mentioned above. Although each of these factors globally affected trans-splicing, the major effect on mRNA levels resulted from the role of these splicing factors in regulating mRNA stability. Biochemical purification of these basal splicing factors identified their association with RNA-binding proteins that are implicated in functions other than splicing. We propose that the binding of splicing factors to the 3′ UTR affects the stability of mRNAs, and we demonstrate this concept using a mini-gene assay, showing that U2AF65 binds a PPT site present in the 3′ UTR. We propose that trans-splicing, known to be coupled to polyadenylation (
      • Hug M.
      • Hotz H.R.
      • Hartmann C.
      • Clayton C.
      Hierarchies of RNA-processing signals in a trypanosome surface antigen mRNA precursor.
      ,
      • LeBowitz J.H.
      • Smith H.Q.
      • Rusche L.
      • Beverley S.M.
      Coupling of poly(A) site selection and trans-splicing in Leishmania.
      ,
      • Matthews K.R.
      • Tschudi C.
      • Ullu E.
      A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes.
      ,
      • Vassella E.
      • Braun R.
      • Roditi I.
      Control of polyadenylation and alternative splicing of transcripts from adjacent genes in a procyclin expression site. A dual role for polypyrimidine tracts in trypanosomes?.
      ), determines which poly(A) site will be chosen and thus the size of the 3′ UTR and its ability to interact with basal splicing factors and other binding proteins that dictate mRNA stability.

      DISCUSSION

      The study identifies novel and unconventional roles for basal splicing factors in trypanosomes. All the splicing factors studied, U2AF65, SF1, and U2AF35, were shown to affect both trans-splicing and mRNA stability. The involvement of these factors in mRNA stability is based on the following: 1) up-regulation of transcripts in the silenced cells even before the detection of splicing defects; 2) localization of splicing factors also in the cytoplasm; 3) mRNA decay assay demonstrating changes in half-life during silencing; 4) co-purification of splicing factors with proteins implicated in mRNA stability; 5) mature mRNA were shown to bind to the splicing factors; and 6) studies using a mini-gene demonstrating that mutating a PPT at the 3′ UTR affects the stability of the mRNA. Despite the direct interaction of splicing factors with mature mRNAs, we cannot at this point exclude the possibility that the transcriptome under these depletions may also be influenced by a change in key master regulators that exert an effect on their target genes.
      We propose that splicing factors bind to splicing signals present in the 3′ UTR of target genes, many of which undergo differential polyadenylation. Indeed, transcripts regulated by U2AF65 and SF1 were shown to have higher polyadenylation heterogeneity (Table 1). In addition, transcripts regulated by splicing factors are enriched with transcripts recently shown to undergo differential polyadenylation (
      • Kolev N.G.
      • Franklin J.B.
      • Carmi S.
      • Shi H.
      • Michaeli S.
      • Tschudi C.
      The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.
      ). Our data suggest that different splicing factors control different subsets of biological functions, including metabolic enzymes, chaperones, transporters, RNA processing, signaling factors, and proteins involved in movement and trafficking. Many of these regulated genes were shown to be differentially regulated during cycling between the two hosts. Our data support the existence of RNA operons whose co-expression dictates the transcriptome during parasite differentiation (
      • Queiroz R.
      • Benz C.
      • Fellenberg K.
      • Hoheisel J.D.
      • Clayton C.
      Transcriptome analysis of differentiating trypanosomes reveals the existence of multiple post-transcriptional regulons.
      ,
      • Fernández-Moya S.M.
      • Estévez A.M.
      Post-transcriptional control and the role of RNA-binding proteins in gene regulation in trypanosomatid protozoan parasites.
      ).

      Are All Splicing Factors Important for Trans-splicing of Each mRNA?

      The most surprising result presented in this study is that the down-regulation of splicing factors by RNAi resulted in up-regulation of selected mRNAs, suggesting a very dominant role of these factors in regulating the stability of mRNAs in these parasites. In a similar study in mammalian cells, where the transcriptome following U2AF35 and U2AF65 depletion by RNAi was examined, it was found that the majority of the mRNAs were down-regulated (
      • Gama-Carvalho M.
      • Barbosa-Morais N.L.
      • Brodsky A.S.
      • Silver P.A.
      • Carmo-Fonseca M.
      Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors.
      ,
      • Pacheco T.R.
      • Moita L.F.
      • Gomes A.Q.
      • Hacohen N.
      • Carmo-Fonseca M.
      RNA interference knockdown of hU2AF35 impairs cell cycle progression and modulates alternative splicing of Cdc25 transcripts.
      ), suggesting that the dominant role of splicing factors on mRNA stability is unique to trypanosomes. This might be due to the very special mechanisms that regulate trypanosome gene expression and obligate linkage between trans-splicing and polyadenylation, giving rise to 3′ UTRs that contain splicing signals that can bind splicing factors (
      • Michaeli S.
      Trans-splicing in trypanosomes. Machinery and its impact on the parasite transcriptome.
      ).
      This unique feature explains how mRNAs can be elevated in trypanosomes during silencing of major splicing factors. Such regulated transcripts most probably bind splicing factors that, under normal conditions, destabilize the mature mRNAs. Splicing defects detected by accumulation of precursors were observed only after 4–5 days of silencing (Fig. 2), whereas the effect on stability was already observed at the 2nd day of silencing.
      Although the effect of splicing factor depletion on splicing is masked by its effect on mRNA stability, these factors are clearly required for trans-splicing (Fig. 1). Inhibition of trans-splicing did contribute significantly to the results observed in this study, because all depletions affected the Y structure intermediate. However, differential effects on transcripts were observed that are significantly correlated with the splicing signals of the genes (Table 1). Indeed, among the transcripts down-regulated in U2AF65-depleted cells are transcripts with short PPT that are likely to be affected more severely when this factor is limited. In addition, transcripts down-regulated by U2AF35 have low splice site diversity, suggesting that these transcripts are more reliant on the presence of U2AF35. This may suggest that not all transcripts rely on all factors for their splicing. This was also shown in other systems such as yeast, Drosophila, and mammals. In mammals, the depletion of U2AF65 mainly affects transcripts with a weak 3′ splice site (
      • Pacheco T.R.
      • Coelho M.B.
      • Desterro J.M.
      • Mollet I.
      • Carmo-Fonseca M.
      In vivo requirement of the small subunit of U2AF for recognition of a weak 3′ splice site.
      ). Moreover, U2AF35 silencing in mammals leads to reduction in the level of only 400 transcripts and not of the entire transcriptome (
      • Pacheco T.R.
      • Moita L.F.
      • Gomes A.Q.
      • Hacohen N.
      • Carmo-Fonseca M.
      RNA interference knockdown of hU2AF35 impairs cell cycle progression and modulates alternative splicing of Cdc25 transcripts.
      ). In yeast, depletion studies on 18 different mRNA processing factors revealed groups of genes with different degrees of dependence on these factors (
      • Clark T.A.
      • Sugnet C.W.
      • Ares Jr., M.
      Genome-wide analysis of mRNA processing in yeast using splicing-specific microarrays.
      ). Combined data from this study and from the studies in mammals and yeast suggest that transcripts may differ in their need for basal splicing factors.
      It should be noted that this study only addresses cases in which the depletion severely affected splicing, mRNA stability, or both. Thus, the full extent of changes in the transcriptome is far more complex. For instance, there could be cases, not detected in our experiments, in which the level of the mature mRNA did not change because the splicing led to a decrease in the transcript, but mRNA stability increased its level, thus leading to no net change in the level of mRNA. Indeed, our results revealed changes in only a few hundred genes in each depletion and not in all ∼9000 trans-spliced transcripts present in the T. brucei genome. RNA-seq analysis of the transcriptome of the splicing-depleted factors studied here should yield a more quantitative picture, demonstrating effects on both mature and intergenic precursors.

      Involvement of Splicing Factors in mRNA Stability and Translation

      The involvement of basal splicing factors in functions other than splicing was reported in only a few studies. The splicing factor ASF/SF2, an SR protein, was shown in mammals to regulate the stability of PKC-1 by binding to a regulatory sequence in the 3′ UTR of its transcript (
      • Lemaire R.
      • Prasad J.
      • Kashima T.
      • Gustafson J.
      • Manley J.L.
      • Lafyatis R.
      Stability of a PKCI-1-related mRNA is controlled by the splicing factor ASF/SF2. A novel function for SR proteins.
      ). In addition, mammalian U2AF65 and PTB were shown to bind to mature mRNAs (
      • Gama-Carvalho M.
      • Barbosa-Morais N.L.
      • Brodsky A.S.
      • Silver P.A.
      • Carmo-Fonseca M.
      Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors.
      ). More recent studies used cross-linking and immunoprecipitation to demonstrate that mammalian SF1 binds not only to pre-mRNA at the branch site but also mRNAs spanning exon junctions (
      • Corioni M.
      • Antih N.
      • Tanackovic G.
      • Zavolan M.
      • Krämer A.
      Analysis of in situ pre-mRNA targets of human splicing factor SF1 reveals a function in alternative splicing.
      ). CLIP data also demonstrated the binding of mammalian SF2/ASF to mature mRNA in the cytoplasm (
      • Sanford J.R.
      • Coutinho P.
      • Hackett J.A.
      • Wang X.
      • Ranahan W.
      • Caceres J.F.
      Identification of nuclear and cytoplasmic mRNA targets for the shuttling protein SF2/ASF.
      ). Moreover, the mammalian splicing factor, SFRS1, was shown to bind targets within mRNAs, miRNA, snoRNA, and noncoding RNA (
      • Sanford J.R.
      • Wang X.
      • Mort M.
      • Vanduyn N.
      • Cooper D.N.
      • Mooney S.D.
      • Edenberg H.J.
      • Liu Y.
      Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts.
      ). Most recently, the RNA-binding protein HuR was shown to bind not only to 3′ UTR but also to intronic sequences, and its knockdown suggests its function not only in mRNA stability but also in splicing (
      • Lebedeva S.
      • Jens M.
      • Theil K.
      • Schwanhäusser B.
      • Selbach M.
      • Landthaler M.
      • Rajewsky N.
      Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR.
      ).
      Although the studies cited above suggest binding of splicing factors to mature mRNAs, only one study demonstrated the role of these factors in mRNA stability (
      • Lemaire R.
      • Prasad J.
      • Kashima T.
      • Gustafson J.
      • Manley J.L.
      • Lafyatis R.
      Stability of a PKCI-1-related mRNA is controlled by the splicing factor ASF/SF2. A novel function for SR proteins.
      ). In contrast, the effect observed in this study regarding the role of basal splicing factors on mRNA stability is very profound and significant. Recently, it was suggested that certain factors function as master coordinators of gene expression. One such complex is yeast Rbp4/6, which is part of the polymerase II complex but also represents a class of mRNA coordinators that integrate the various stages of gene expression (
      • Harel-Sharvit L.
      • Eldad N.
      • Haimovich G.
      • Barkai O.
      • Duek L.
      • Choder M.
      RNA polymerase II subunits link transcription and mRNA decay to translation.
      ). The trypanosome factor U2AF35 controls splicing, mRNA decay, and possibly translation and thus may serve as such coordinators of gene expression in these parasites.
      The factors known to regulate mRNA stability in trypanosomes are proteins carrying RRMs, proteins containing a CCH type zinc finger, and PUF proteins (
      • Kramer S.
      • Carrington M.
      Trans-acting proteins regulating mRNA maturation, stability, and translation in trypanosomatids.
      ). Each of the RRM-containing proteins seems to affect the stability (stabilization or destabilization) of a different subset of proteins. These cumulative data suggest the existence of RNA operons, i.e. genes that participate in a single pathway or biological function that are co-regulated by a common factor. The presence of RNA operons in T. brucei gene expression was also noted in a study that determined the transcriptome during differentiation and demonstrated that ribosomal proteins, flagellar biogenesis proteins, and factors from the same metabolic pathways are often co-regulated (
      • Queiroz R.
      • Benz C.
      • Fellenberg K.
      • Hoheisel J.D.
      • Clayton C.
      Transcriptome analysis of differentiating trypanosomes reveals the existence of multiple post-transcriptional regulons.
      ). Our study also supports the operon type of regulation in the trypanosome transcriptome. Over 20% of the transcripts regulated by U2AF65/SF1 are metabolic enzymes. Interestingly, many of the genes regulated by these splicing factors are transcripts whose expression is changed during the differentiation of the parasites (supplemental S-3). As stated above, we cannot exclude the possibility that some of the effects observed on the transcriptome emerge from a perturbation in expression of a master regulator(s), also controlling the stability of many mRNAs.

      How Could a Single Factor Both Stabilize and Destabilize mRNAs?

      The mRNAs that are destabilized during factor silencing may carry hypersensitive sites that are protected by the factor. Conversely, those that are stabilized during silencing may be recruited by the factor to the degradation machinery. But how can the same protein be recruited for such different machineries? This depends solely on the other factors that bound the mRNA. The combinatorial binding of other binding proteins may generate a multitude of complexes each formed under different physiological and developmental conditions. Indeed, a variety of RNA-binding proteins co-purified with these splicing factors; these proteins can either directly interact with the factor or, more likely, interact with the transcripts bound by the factor. Interestingly, U2AF65- and SF1-regulated transcripts are also rich in purine-rich exons, suggesting that regulated transcripts are bound by several different factors.

      Purification of Complexes Containing the Splicing Factors Supports Their Role in mRNA Decay and Translation

      Our mass spectrometry data identified several RNA-binding proteins that may also function in regulating mRNA stability and even translational control. The factors examined in this study are known for their splicing activity. However, the majority of proteins associated with these splicing factors are implicated in mRNA stability, ribosome function, and biogenesis (especially those associated with U2AF35). Spliceosomal complexes were comprehensively identified by purifying Sm proteins from both T. brucei (
      • Luz Ambrósio D.
      • Lee J.H.
      • Panigrahi A.K.
      • Nguyen T.N.
      • Cicarelli R.M.
      • Günzl A.
      Spliceosomal proteomics in Trypanosoma brucei reveal new RNA splicing factors.
      ,
      • Palfi Z.
      • Jaé N.
      • Preusser C.
      • Kaminska K.H.
      • Bujnicki J.M.
      • Lee J.H.
      • Günzl A.
      • Kambach C.
      • Urlaub H.
      • Bindereif A.
      SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes.
      ) and Leishmania (
      • Tkacz I.D.
      • Gupta S.K.
      • Volkov V.
      • Romano M.
      • Haham T.
      • Tulinski P.
      • Lebenthal I.
      • Michaeli S.
      Analysis of spliceosomal proteins in trypanosomatids reveals novel functions in mRNA processing.
      ). In yeast, spliceosomal complexes were also identified by tagging Sm or snRNP-specific complexes (
      • Jurica M.S.
      • Moore M.J.
      Pre-mRNA splicing. Awash in a sea of proteins.
      ). This study failed to detect components of the spliceosomal complex, including core proteins such as Sm proteins, and U snRNP-specific proteins. These results may indicate either that the spliceosomal complexes carrying the studied factors are fragile and dissociate during purification or that the level of spliceosomal complexes containing these factors is low compared with sub-spliceosomal complexes that include these factors. Indeed, studies in mammals identified a large pool of extra-spliceosomal complexes containing U2AF65 and SF1 (
      • Rino J.
      • Desterro J.M.
      • Pacheco T.R.
      • Gadella Jr., T.W.
      • Carmo-Fonseca M.
      Splicing factors SF1 and U2AF associate in extra spliceosomal complexes.
      ).

      Mechanism by Which Splicing Factors Affect mRNA Stability in Trypanosomes

      By what mechanism could splicing factors bind so extensively to sequences other than the splicing signals present on mRNAs? In trypanosomes, such interactions might be prevalent due to two major specific characteristics of gene expression. First, extensive alternative polyadenylation was observed, with over 400 cases already described in the procyclic stage (
      • Kolev N.G.
      • Franklin J.B.
      • Carmi S.
      • Shi H.
      • Michaeli S.
      • Tschudi C.
      The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.
      ). Indeed, U2AF65- and SF1-regulated transcripts were shown in this study to have higher polyadenylation heterogeneity and to be enriched with transcripts previously shown to undergo differential polyadenylation (
      • Kolev N.G.
      • Franklin J.B.
      • Carmi S.
      • Shi H.
      • Michaeli S.
      • Tschudi C.
      The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.
      ). Second, trans-splicing and polyadenylation are linked (
      • Hug M.
      • Hotz H.R.
      • Hartmann C.
      • Clayton C.
      Hierarchies of RNA-processing signals in a trypanosome surface antigen mRNA precursor.
      ,
      • LeBowitz J.H.
      • Smith H.Q.
      • Rusche L.
      • Beverley S.M.
      Coupling of poly(A) site selection and trans-splicing in Leishmania.
      ,
      • Matthews K.R.
      • Tschudi C.
      • Ullu E.
      A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes.
      ,
      • Vassella E.
      • Braun R.
      • Roditi I.
      Control of polyadenylation and alternative splicing of transcripts from adjacent genes in a procyclin expression site. A dual role for polypyrimidine tracts in trypanosomes?.
      ). In trypanosomes, trans-splicing determines which poly(A) site will be selected and hence the size of the 3′ UTR; this choice in turn dictates the stability of the mRNA due to the presence of binding sites for a variety of RNA-binding proteins. However, orphan splicing signals that are not coupled to polyadenylation also may exist at the 3′ UTR of genes and serve as binding sites that exert their effect on mature mRNAs.
      Regulation of mRNA stability by splicing factors in trypanosomes might be general and not trypanosome-specific, and splicing factors may also affect the stability of mRNA by binding to the 3′ UTR in other systems. Indeed, support for this notion was recently provided for mammalian SF1, which was shown to bind the 3′ UTR of mammalian genes (
      • Corioni M.
      • Antih N.
      • Tanackovic G.
      • Zavolan M.
      • Krämer A.
      Analysis of in situ pre-mRNA targets of human splicing factor SF1 reveals a function in alternative splicing.
      ). Thus, the role of basal splicing factors in mRNA stability, suggested for the first time in this study, might operate in other systems as well. Although the data presented in this study support the direct binding of the splicing factor to mature mRNA, most probably to the 3′ UTR, we must also consider the possibility that the mRNA stability changes observed may stem not only from the direct binding of the splicing factors to the 3′ UTR but may result from secondary perturbations. For instance, the effect on stability may result from perturbation induced by splicing defect of a factor(s) involved in mRNA stability. Extensive genome-wide CLIP-Seq assays are expected to differentiate between direct binding and secondary effects.
      In sum, this study highlights the contribution of splicing factors not only to the efficiency of trans-splicing but also to mRNA stability, which seems to be the most robust process determining the parasite transcriptome. The decision regarding the choice of splicing and polyadenylation signals determines the composition of the 3′ UTR, and thus the half-life of the target mRNA. This regulation is most probably pivotal for the adaptation of gene expression during the developmental cycle of the parasite and for responding to external or internal stimuli.

      Acknowledgments

      We thank Dr. Mark Katzenellenbogen for the initial analysis of microarray data and Asher Pivko for contributions to the early stages of the project.

      REFERENCES

        • Agabian N.
        Trans-splicing of nuclear pre-mRNAs.
        Cell. 1990; 61: 1157-1160
        • Liang X.H.
        • Haritan A.
        • Uliel S.
        • Michaeli S.
        Trans- and cis-splicing in trypanosomatids. Mechanism, factors, and regulation.
        Eukaryote Cell. 2003; 2: 830-840
        • Michaeli S.
        Trans-splicing in trypanosomes. Machinery and its impact on the parasite transcriptome.
        Future Microbiol. 2011; 6: 459-474
        • Kolev N.G.
        • Franklin J.B.
        • Carmi S.
        • Shi H.
        • Michaeli S.
        • Tschudi C.
        The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.
        PLoS Pathog. 2010; 6: e1001090
        • Siegel T.N.
        • Hekstra D.R.
        • Wang X.
        • Dewell S.
        • Cross G.A.
        Genome-wide analysis of mRNA abundance in two life-cycle stages of Trypanosoma brucei and identification of splicing and polyadenylation sites.
        Nucleic Acids Res. 2010; 38: 4946-4957
        • Nilsson D.
        • Gunasekera K.
        • Mani J.
        • Osteras M.
        • Farinelli L.
        • Baerlocher L.
        • Roditi I.
        • Ochsenreiter T.
        Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei.
        PLoS Pathog. 2010; 6: e1001037
        • Kramer S.
        • Carrington M.
        Trans-acting proteins regulating mRNA maturation, stability, and translation in trypanosomatids.
        Trends Parasitol. 2011; 27: 23-30
        • Clayton C.
        • Shapira M.
        Post-transcriptional regulation of gene expression in trypanosomes and leishmanias.
        Mol. Biochem. Parasitol. 2007; 156: 93-101
        • Günzl A.
        The pre-mRNA splicing machinery of trypanosomes. Complex or simplified?.
        Eukaryot. Cell. 2010; 9: 1159-1170
        • Vazquez M.P.
        • Mualem D.
        • Bercovich N.
        • Stern M.Z.
        • Nyambega B.
        • Barda O.
        • Nasiga D.
        • Gupta S.K.
        • Michaeli S.
        • Levin M.J.
        Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65, and SF1.
        Mol. Biochem. Parasitol. 2009; 164: 137-146
        • Vázquez M.
        • Atorrasagasti C.
        • Bercovich N.
        • Volcovich R.
        • Levin M.J.
        Unique features of the Trypanosoma cruzi U2AF35 splicing factor.
        Mol. Biochem. Parasitol. 2003; 128: 77-81
        • Kol G.
        • Lev-Maor G.
        • Ast G.
        Human-mouse comparative analysis reveals that branch-site plasticity contributes to splicing regulation.
        Hum. Mol. Genet. 2005; 14: 1559-1568
        • Schwartz S.H.
        • Silva J.
        • Burstein D.
        • Pupko T.
        • Eyras E.
        • Ast G.
        Large scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes.
        Genome Res. 2008; 18: 88-103
        • Ismaïli N.
        • Pérez-Morga D.
        • Walsh P.
        • Cadogan M.
        • Pays A.
        • Tebabi P.
        • Pays E.
        Characterization of a Trypanosoma brucei SR domain-containing protein bearing homology to cis-spliceosomal U1 70-kDa proteins.
        Mol. Biochem. Parasitol. 2000; 106: 109-120
        • Ismaïli N.
        • Pérez-Morga D.
        • Walsh P.
        • Mayeda A.
        • Pays A.
        • Tebabi P.
        • Krainer A.R.
        • Pays E.
        Characterization of a SR protein from Trypanosoma brucei with homology to RNA-binding cis-splicing proteins.
        Mol. Biochem. Parasitol. 1999; 102: 103-115
        • Manger I.D.
        • Boothroyd J.C.
        Identification of a nuclear protein in Trypanosoma brucei with homology to RNA-binding proteins from cis-splicing systems.
        Mol. Biochem. Parasitol. 1998; 97: 1-11
        • Estévez A.M.
        The RNA-binding protein TbDRBD3 regulates the stability of a specific subset of mRNAs in trypanosomes.
        Nucleic Acids Res. 2008; 36: 4573-4586
        • Stern M.Z.
        • Gupta S.K.
        • Salmon-Divon M.
        • Haham T.
        • Barda O.
        • Levi S.
        • Wachtel C.
        • Nilsen T.W.
        • Michaeli S.
        Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism.
        RNA. 2009; 15: 648-665
        • Hug M.
        • Hotz H.R.
        • Hartmann C.
        • Clayton C.
        Hierarchies of RNA-processing signals in a trypanosome surface antigen mRNA precursor.
        Mol. Cell. Biol. 1994; 14: 7428-7435
        • LeBowitz J.H.
        • Smith H.Q.
        • Rusche L.
        • Beverley S.M.
        Coupling of poly(A) site selection and trans-splicing in Leishmania.
        Genes Dev. 1993; 7: 996-1007
        • Matthews K.R.
        • Tschudi C.
        • Ullu E.
        A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes.
        Genes Dev. 1994; 8: 491-501
        • Vassella E.
        • Braun R.
        • Roditi I.
        Control of polyadenylation and alternative splicing of transcripts from adjacent genes in a procyclin expression site. A dual role for polypyrimidine tracts in trypanosomes?.
        Nucleic Acids Res. 1994; 22: 1359-1364
        • Mandelboim M.
        • Barth S.
        • Biton M.
        • Liang X.H.
        • Michaeli S.
        Silencing of Sm proteins in Trypanosoma brucei by RNA interference captured a novel cytoplasmic intermediate in spliced leader RNA biogenesis.
        J. Biol. Chem. 2003; 278: 51469-51478
        • Aphasizhev R.
        • Aphasizheva I.
        • Nelson R.E.
        • Gao G.
        • Simpson A.M.
        • Kang X.
        • Falick A.M.
        • Sbicego S.
        • Simpson L.
        Isolation of a U-insertion/deletion editing complex from Leishmania tarentolae mitochondria.
        EMBO J. 2003; 22: 913-924
        • Palfi Z.
        • Schimanski B.
        • Günzl A.
        • Lücke S.
        • Bindereif A.
        U1 small nuclear RNP from Trypanosoma brucei. A minimal U1 snRNA with unusual protein components.
        Nucleic Acids Res. 2005; 33: 2493-2503
        • Ellis J.A.
        • Fish W.R.
        • Sileghem M.
        • McOdimba F.
        A colorimetric assay for trypanosome viability and metabolic function.
        Vet. Parasitol. 1993; 50: 143-149
        • Tkacz I.D.
        • Gupta S.K.
        • Volkov V.
        • Romano M.
        • Haham T.
        • Tulinski P.
        • Lebenthal I.
        • Michaeli S.
        Analysis of spliceosomal proteins in trypanosomatids reveals novel functions in mRNA processing.
        J. Biol. Chem. 2010; 285: 27982-27999
        • Hury A.
        • Goldshmidt H.
        • Tkacz I.D.
        • Michaeli S.
        Trypanosome spliced-leader-associated RNA (SLA1) localization and implications for spliced-leader RNA biogenesis.
        Eukaryot. Cell. 2009; 8: 56-68
        • Liang X.H.
        • Liu L.
        • Michaeli S.
        Identification of the first trypanosome H/ACA RNA that guides pseudouridine formation on rRNA.
        J. Biol. Chem. 2001; 276: 40313-40318
        • Xu Y.
        • Liu L.
        • Lopez-Estraño C.
        • Michaeli S.
        Expression studies on clustered trypanosomatid box C/D small nucleolar RNAs.
        J. Biol. Chem. 2001; 276: 14289-14298
        • Gupta S.K.
        • Hury A.
        • Ziporen Y.
        • Shi H.
        • Ullu E.
        • Michaeli S.
        Small nucleolar RNA interference in Trypanosoma brucei. Mechanism and utilization for elucidating the function of snoRNAs.
        Nucleic Acids Res. 2010; 38: 7236-7247
        • Yang Y.H.
        • Dudoit S.
        • Luu P.
        • Lin D.M.
        • Peng V.
        • Ngai J.
        • Speed T.P.
        Normalization for cDNA microarray data. A robust composite method addressing single and multiple slide systematic variation.
        Nucleic Acids Res. 2002; 30: e15
        • Michaeli S.
        • Roberts T.G.
        • Watkins K.P.
        • Agabian N.
        Isolation of distinct small ribonucleoprotein particles containing the spliced leader and U2 RNAs of Trypanosoma brucei.
        J. Biol. Chem. 1990; 265: 10582-10588
        • Siegel T.N.
        • Tan K.S.
        • Cross G.A.
        Systematic study of sequence motifs for RNA trans splicing in Trypanosoma brucei.
        Mol. Cell. Biol. 2005; 25: 9586-9594
        • Schimanski B.
        • Nguyen T.N.
        • Günzl A.
        Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination.
        Eukaryot. Cell. 2005; 4: 1942-1950
        • Jaé N.
        • Wang P.
        • Gu T.
        • Hühn M.
        • Palfi Z.
        • Urlaub H.
        • Bindereif A.
        Essential role of a trypanosome U4-specific Sm core protein in small nuclear ribonucleoprotein assembly and splicing.
        Eukaryot. Cell. 2010; 9: 379-386
        • Liang X.H.
        • Liu Q.
        • Liu L.
        • Tschudi C.
        • Michaeli S.
        Analysis of spliceosomal complexes in Trypanosoma brucei and silencing of two splicing factors Prp31 and Prp43.
        Mol. Biochem. Parasitol. 2006; 145: 29-39
        • Luz Ambrósio D.
        • Lee J.H.
        • Panigrahi A.K.
        • Nguyen T.N.
        • Cicarelli R.M.
        • Günzl A.
        Spliceosomal proteomics in Trypanosoma brucei reveal new RNA splicing factors.
        Eukaryot. Cell. 2009; 8: 990-1000
        • Ruan J.P.
        • Shen S.
        • Ullu E.
        • Tschudi C.
        Evidence for a capping enzyme with specificity for the trypanosome spliced leader RNA.
        Mol Biochem. Parasitol. 2007; 156: 246-254
        • Tkacz I.D.
        • Cohen S.
        • Salmon-Divon M.
        • Michaeli S.
        Identification of the heptameric Lsm complex that binds U6 snRNA in Trypanosoma brucei.
        Mol. Biochem. Parasitol. 2008; 160: 22-31
        • Tkacz I.D.
        • Lustig Y.
        • Stern M.Z.
        • Biton M.
        • Salmon-Divon M.
        • Das A.
        • Bellofatto V.
        • Michaeli S.
        Identification of novel snRNA-specific Sm proteins that bind selectively to U2 and U4 snRNAs in Trypanosoma brucei.
        RNA. 2007; 13: 30-43
        • Dennis Jr., G.
        • Sherman B.T.
        • Hosack D.A.
        • Yang J.
        • Gao W.
        • Lane H.C.
        • Lempicki R.A.
        DAVID: Database for annotation, visualization, and integrated discovery.
        Genome Biol. 2003; 4: P3
        • Queiroz R.
        • Benz C.
        • Fellenberg K.
        • Hoheisel J.D.
        • Clayton C.
        Transcriptome analysis of differentiating trypanosomes reveals the existence of multiple post-transcriptional regulons.
        BMC Genomics. 2009; 10: 495
        • Archer S.K.
        • Inchaustegui D.
        • Queiroz R.
        • Clayton C.
        The cell cycle regulated transcriptome of Trypanosoma brucei.
        PLoS ONE. 2011; 6: e18425
        • Jensen B.C.
        • Sivam D.
        • Kifer C.T.
        • Myler P.J.
        • Parsons M.
        Widespread variation in transcript abundance within and across developmental stages of Trypanosoma brucei.
        BMC Genomics. 2009; 10: 482
        • Kabani S.
        • Fenn K.
        • Ross A.
        • Ivens A.
        • Smith T.K.
        • Ghazal P.
        • Matthews K.
        Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei.
        BMC Genomics. 2009; 10: 427
        • Colasante C.
        • Robles A.
        • Li C.H.
        • Schwede A.
        • Benz C.
        • Voncken F.
        • Guilbride D.L.
        • Clayton C.
        Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei.
        Mol. Biochem. Parasitol. 2007; 151: 193-204
        • Manful T.
        • Fadda A.
        • Clayton C.
        The role of the 5′–3′ exoribonuclease XRNA in transcriptome-wide mRNA degradation.
        RNA. 2011; 17: 2039-2047
        • Anderson P.
        • Kedersha N.
        Visibly stressed. The role of eIF2, TIA-1, and stress granules in protein translation.
        Cell Stress Chaperones. 2002; 7: 213-221
        • De Gaudenzi J.
        • Frasch A.C.
        • Clayton C.
        RNA-binding domain proteins in kinetoplastids. A comparative analysis.
        Eukaryot. Cell. 2005; 4: 2106-2114
        • Wurst M.
        • Robles A.
        • Po J.
        • Luu V.D.
        • Brems S.
        • Marentije M.
        • Stoitsova S.
        • Quijada L.
        • Hoheisel J.
        • Stewart M.
        • Hartmann C.
        • Clayton C.
        An RNAi screen of the RRM-domain proteins of.
        Trypanosoma brucei. Mol. Biochem. Parasitol. 2009; 163: 61-65
        • Archer S.K.
        • Luu V.D.
        • de Queiroz R.A.
        • Brems S.
        • Clayton C.
        Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle.
        PLoS Pathog. 2009; 5: e1000565
        • Droll D.
        • Archer S.
        • Fenn K.
        • Delhi P.
        • Matthews K.
        • Clayton C.
        The trypanosome Pumilio-domain protein PUF7 associates with a nuclear cyclophilin and is involved in ribosomal RNA maturation.
        FEBS Lett. 2010; 584: 1156-1162
        • Hartmann C.
        • Benz C.
        • Brems S.
        • Ellis L.
        • Luu V.D.
        • Stewart M.
        • D'Orso I.
        • Busold C.
        • Fellenberg K.
        • Frasch A.C.
        • Carrington M.
        • Hoheisel J.
        • Clayton C.E.
        Small trypanosome RNA-binding proteins TbUBP1 and TbUBP2 influence expression of F-box protein mRNAs in bloodstream trypanosomes.
        Eukaryot. Cell. 2007; 6: 1964-1978
        • Hartmann C.
        • Clayton C.
        Regulation of a transmembrane protein gene family by the small RNA-binding proteins TbUBP1 and TbUBP2.
        Mol. Biochem. Parasitol. 2008; 157: 112-115
        • Barth S.
        • Hury A.
        • Liang X.H.
        • Michaeli S.
        Elucidating the role of H/ACA-like RNAs in trans-splicing and rRNA processing via RNA interference silencing of the Trypanosoma brucei CBF5 pseudouridine synthase.
        J. Biol. Chem. 2005; 280: 34558-34568
        • Barth S.
        • Shalem B.
        • Hury A.
        • Tkacz I.D.
        • Liang X.H.
        • Uliel S.
        • Myslyuk I.
        • Doniger T.
        • Salmon-Divon M.
        • Unger R.
        • Michaeli S.
        Elucidating the role of C/D snoRNA in rRNA processing and modification in Trypanosoma brucei.
        Eukaryot. Cell. 2008; 7: 86-101
        • Jensen B.C.
        • Wang Q.
        • Kifer C.T.
        • Parsons M.
        The NOG1 GTP-binding protein is required for biogenesis of the 60 S ribosomal subunit.
        J. Biol. Chem. 2003; 278: 32204-32211
        • Prohaska K.
        • Williams N.
        Assembly of the Trypanosoma brucei 60 S ribosomal subunit nuclear export complex requires trypanosome-specific proteins P34 and P37.
        Eukaryot. Cell. 2009; 8: 77-87
        • Alves L.R.
        • Avila A.R.
        • Correa A.
        • Holetz F.B.
        • Mansur F.C.
        • Manque P.A.
        • de Menezes J.P.
        • Buck G.A.
        • Krieger M.A.
        • Goldenberg S.
        Proteomic analysis reveals the dynamic association of proteins with translated mRNAs in Trypanosoma cruzi.
        Gene. 2010; 452: 72-78
        • Fernández-Moya S.M.
        • Estévez A.M.
        Post-transcriptional control and the role of RNA-binding proteins in gene regulation in trypanosomatid protozoan parasites.
        Wiley Interdiscip. Rev. RNA. 2010; 1: 34-46
        • Gama-Carvalho M.
        • Barbosa-Morais N.L.
        • Brodsky A.S.
        • Silver P.A.
        • Carmo-Fonseca M.
        Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors.
        Genome Biol. 2006; 7: R113
        • Pacheco T.R.
        • Moita L.F.
        • Gomes A.Q.
        • Hacohen N.
        • Carmo-Fonseca M.
        RNA interference knockdown of hU2AF35 impairs cell cycle progression and modulates alternative splicing of Cdc25 transcripts.
        Mol. Biol. Cell. 2006; 17: 4187-4199
        • Pacheco T.R.
        • Coelho M.B.
        • Desterro J.M.
        • Mollet I.
        • Carmo-Fonseca M.
        In vivo requirement of the small subunit of U2AF for recognition of a weak 3′ splice site.
        Mol. Cell. Biol. 2006; 26: 8183-8190
        • Clark T.A.
        • Sugnet C.W.
        • Ares Jr., M.
        Genome-wide analysis of mRNA processing in yeast using splicing-specific microarrays.
        Science. 2002; 296: 907-910
        • Lemaire R.
        • Prasad J.
        • Kashima T.
        • Gustafson J.
        • Manley J.L.
        • Lafyatis R.
        Stability of a PKCI-1-related mRNA is controlled by the splicing factor ASF/SF2. A novel function for SR proteins.
        Genes Dev. 2002; 16: 594-607
        • Corioni M.
        • Antih N.
        • Tanackovic G.
        • Zavolan M.
        • Krämer A.
        Analysis of in situ pre-mRNA targets of human splicing factor SF1 reveals a function in alternative splicing.
        Nucleic Acids Res. 2011; 39: 1868-1879
        • Sanford J.R.
        • Coutinho P.
        • Hackett J.A.
        • Wang X.
        • Ranahan W.
        • Caceres J.F.
        Identification of nuclear and cytoplasmic mRNA targets for the shuttling protein SF2/ASF.
        PLoS ONE. 2008; 3: e3369
        • Sanford J.R.
        • Wang X.
        • Mort M.
        • Vanduyn N.
        • Cooper D.N.
        • Mooney S.D.
        • Edenberg H.J.
        • Liu Y.
        Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts.
        Genome Res. 2009; 19: 381-394
        • Lebedeva S.
        • Jens M.
        • Theil K.
        • Schwanhäusser B.
        • Selbach M.
        • Landthaler M.
        • Rajewsky N.
        Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR.
        Mol. Cell. 2011; 43: 340-352
        • Harel-Sharvit L.
        • Eldad N.
        • Haimovich G.
        • Barkai O.
        • Duek L.
        • Choder M.
        RNA polymerase II subunits link transcription and mRNA decay to translation.
        Cell. 2010; 143: 552-563
        • Palfi Z.
        • Jaé N.
        • Preusser C.
        • Kaminska K.H.
        • Bujnicki J.M.
        • Lee J.H.
        • Günzl A.
        • Kambach C.
        • Urlaub H.
        • Bindereif A.
        SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes.
        Genes Dev. 2009; 23: 1650-1664
        • Jurica M.S.
        • Moore M.J.
        Pre-mRNA splicing. Awash in a sea of proteins.
        Mol. Cell. 2003; 12: 5-14
        • Rino J.
        • Desterro J.M.
        • Pacheco T.R.
        • Gadella Jr., T.W.
        • Carmo-Fonseca M.
        Splicing factors SF1 and U2AF associate in extra spliceosomal complexes.
        Mol. Cell. Biol. 2008; 28: 3045-3057