Alternative splicing is an important cellular mechanism. It allows to produce multiple protein isoforms from a limited number of genes. Regulation of alternative splicing involves cis-acting elements on pre-mRNA and trans-acting splicing factors (SR and hnRNP proteins). Because splicing occurs co-transcriptionaly, chromatin structure appears to have a role in the regulation of alternative splicing. We have studied the effect of histone acetylation on alternative splicing. We have prepared splicing reporter for alternative EDB exon, which is part of the fibronectin gene. We have shown, that the inhibition of histone deacetylases affects splicing pattern of EDB exon from the reporter in the same way as the splicing of the endogenous EDB exon. Furthermore, we have shown, that the structure of the promoter affects splicing of alternative EDB exon from splicing reporter. Currently we have found out, that the structure of the promoter influences the degree of histone H4 acetylation. Inclusion of alternative EDB exon in mRNA was inversely proportional to histon acetylation on the reporter. This work might explain why various promoters have different splicing patterns of alternative exons.

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Gene expression in the eukaryotic cell is a fundamental cellular process, which consists of several distinct steps but extensively coupled to each other. From site of transcription in the nucleus to the cytoplasm, both mRNA and rRNA are associated with a proper set of proteins. These proteins influence RNA processing, transport as well as ribosome maturation. We have tried to take advantage of different model systems to understand the process of eukaryotic gene expression at the post-transcription level. To this end, we have focused on identification and characterization of several specific proteins in the context of mRNP and rRNP particles. We have characterized a novel yeast gene MRD1, which encodes a protein with five RNA-binding domains (RBDs) and is essential for viability. Mrd1p is present in the nucleolus and the nucleoplasm. Depletion of Mrd1p leads to a decrease in the synthesis of 18S rRNA and 40S ribosomal subunits. Mrd1p associates with the 35S prerRNA and the U3 snoRNA and is required for the initial processing of pre-rRNA at the A0-A2 sites. The presence of five RBDs in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage. Meanwhile, an MRD1 homologue, Ct-RBD-1 with six RBDs, has also been identified and shown to involve in ribosome biogenesis in Chironomus tentans. Ct-RBD-1 binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In the cytoplasm, Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that Ct-RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes. We have characterized a novel abundant nucleolar protein, p100 in C. tentans. The p100 protein is located in the fibrillar compartment of the nucleolus, and remains in the nucleolus after digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. These results suggest that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus. We have characterized a Dbp5 homologue in C. tentans, Ct-Dbp5. The protein becomes associated with nascent pre-mRNAs at a large number of active genes, including the Balbiani ring (BR) genes. Ct-Dbp5 is bound to nascent BR pre-mRNP particles and accompanies them through the nucleoplasm and the nuclear pore into the cytoplasm. Nuclear accumulation of Ct-Dbp5 takes place when synthesis and/or export of mRNA are inhibited. Our results indicate that most or all of the shuttling Ct-Dbp5 exiting from the nucleus associated with mRNP. Furthermore, Ct-Dbp5 is present along the mRNP fibril extending into the cytoplasm, supporting the view that Ct-Dbp5 is involved in restructuring the mRNP prior to translation. We have shown that the export receptor CRM1 in C. tentans is associated with BR pre-mRNP while transcription takes place. We have also shown that the GTPase Ran binds to BR pre-mRNP, but its binding mainly in the interchromatin. Although both CRM1 and Ran accompany BR pre-mRNP through the nuclear pore, Leptomycin B treatment reveals that a NES-CRM1-RanGTP complex is not essential for export of the BR mRNP. Our results suggest that several export receptors associate with BR mRNP and that these receptors might have redundant functions in the nuclear export of BR mRNP. We have analyzed four SR proteins, SC35, ASF/SF2, 9G8 and hrp45, in C. tentans. All four SR proteins genes are expressed in salivary gland cells and in several other tissues in a tissue specific pattern. We found that about 90% of all nascent pre-mRNAs bind all four SR proteins, and that approximately 10% of the pre-mRNAs associate with different subsets of the four SR proteins, suggesting that not all of four SR proteins are needed for processing of pre-mRNA. None of three examined SR proteins leave BR pre-mRNP as splicing is completed. Instead, 9G8 accompanies the mRNP to the cytoplasm, while SC35 and hrp45 leave the BR mRNP at the nuclear side of the nuclear pore complex.

Eukaryotic gene expression requires hundreds of proteins and several RNA factors to facilitate nuclear RNA processing. These RNA processing events include RNA transcription, pre-mRNA splicing, pre-ribosomal RNA (pre-rRNA) processing and trafficking of RNA to its proper location in the cell. As we learn more about the molecular details of the factors governing these highly coordinated processes it is becoming increasingly clear that a subset of factors participate in multiple RNA processing pathways to ensure faithful gene expression. Our work completes the characterization of the Abelson pre-mRNA splicing mutants. We have discovered that the prp27-1 splicing mutant is a severe loss of function allele of RAT1, an essential 5’→3’ exoribonuclease. Several alleles of RAT1 have been previously isolated with each conferring an array of phenotypes thus making the elucidation of its essential in vivo function difficult. We set out to determine how mutations within a specific region determines the RNA processing pathway in which Rat1p has been implicated to function within. In our analysis of Rat1p function we discovered the prp27-1 allele exhibits novel 3’ end processing defects never reported in previous rat1 mutants. We performed mutational analysis to examine the coupling of 5’ and 3’ exonucleolytic activities in nuclear RNA processing events. Through our study we have discovered a means by which the cell coordinately regulates the nuclear RNA degradation complexes to ensure efficient processing of pre-RNAs for the faithful execution of eukaryotic gene expression. Additionally, we offer evidence in support of role for Rat1p in promoting mitotic events in vivo. / text

Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.

Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.

Messenger RNAs are transcribed and co-transcriptionally processed in the nucleus, and transported to the cytoplasm. In the cytoplasm, mRNAs serve as the template for protein synthesis and are eventually degraded. The removal of intron sequences from a precursor mRNA is termed splicing and is carried out by the dynamic spliceosome. In this thesis, I describe the regulated splicing of two transcripts in Saccharomyces cerevisiae. I also describe a study where the mechanisms that control the expression of magnesium transporters are elucidated. The pre-mRNA retention and splicing (RES) complex is a spliceosome-associated protein complex that promotes the splicing and nuclear retention of a subset of pre-mRNAs. The RES complex consists of three subunits, Bud13p, Snu17p and Pml1p. We show that the lack of RES factors causes a decrease in the formation of N4-acetylcytidine (ac4C) in tRNAs. This phenotype is caused by inefficient splicing of the pre-mRNA of the TAN1 gene, which is required for the formation of ac4C in tRNAs. The RES mutants also show growth defects that are exacerbated at elevated temperatures. We show that the temperature sensitive phenotype of the bud13Δ and snu17Δ cells is caused by the inefficient splicing of the MED20 pre-mRNA. The MED20 gene encodes a subunit of the Mediator complex. Unspliced pre-mRNAs that enter the cytoplasm are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway, which targets transcripts that contain premature translation termination codons. Consistent with the nuclear retention function of the RES complex, we find that NMD inactivation in the RES mutants leads to the accumulation of both TAN1 and MED20 pre-mRNAs. We also show that the cis-acting elements that promote RES-dependent splicing are different between the TAN1 and MED20 pre-mRNAs. The NMD pathway also targets transcripts with upstream ORFs (uORFs) for degradation. The ALR1 gene encodes the major magnesium importer in yeast, and its expression is controlled by the NMD pathway via a uORF in the 5’ untranslated region. We show that the ribosome reaches the downstream main ORF by a translation reinitiation mechanism. The NMD pathway was shown to control cellular Mg2+ levels by regulating the expression of the ALR1 gene. We further show that the NMD pathway targets the transcripts of the vacuolar Mg2+ exporter Mnr2p and the mitochondrial Mg2+ exporter Mme1p for degradation. In summary, we conclude that the RES complex has a role in the splicing regulation of a subset of transcripts. We also suggest a regulatory role for the NMD pathway in maintaining the cellular Mg2+ concentration by controlling the expression of Mg2+ transporters.

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatio-temporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

Exonic sequences of eukaryotic genes are interspersed with introns which when accurately removed from the primary transcript (pre-mRNA) results in a functional transcript. These splicing reactions are carried out by the spliceosome, consisting of U1, U2, U4, U5, U6 snRNAs and 150 non-snRNP proteins, which assemble onto the pre-mRNA and catalyzes the two invariant transesterification reactions (Will and Luhrmann, 2006). The flexibility in choice of splice sites allows for alternative splicing which has immensely contributed to eukaryotic genome evolution and in diversifying the metazoan proteome (Nilesen and Graveley, 2010). Dynamic yet ordered interactions between U2, U5 and U6 snRNAs and Prp8, Prp16, Prp17, Prp18, Slu7 and Prp22 splicing factors are required in vitro for second-step of splicing of budding yeast and human model transcripts (Umen and Guthrie, 1995a; Horowitz, 2012). ScSlu7 aids 3’ss selection while its strongly associated partner ScPrp18 stabilises U5 snRNA-exonic interactions (James et al., 2002; Aronova et al., 2007). These factors are dispensable in vitro, for the splicing of introns with short branch nucleotide to 3’ss distances (Brys and Schwer, 1996; Zhang and Schwer, 1997). Nearly 43% of fission yeast genes have short introns, with degenerate splice-signals and unconventional Py(n) tracts (Kuhn and Kaufer, 2003). As these features differ extensively from budding yeast and are interestingly more representative of fungal and other eukaryotic introns, fission yeast is an attractive unicellular model to investigate alternate splice-site recognition and assembly mechanisms. Mechanistic details of the second catalytic step are poorly understood in fission yeast. Strikingly, mutations in 3’ss and Py(n) tract intronic cis elements, known to block second step splicing in budding yeast, cause pre-catalytic arrest with unspliced pre-mRNA accumulation in fission yeast (Romfo and Wise, 1997). Studies in our laboratory focussed on understanding the functions for fission yeast SpPrp18 and SpSlu7 predicted to be second-step factors, revealed remarkable differences as compared to their budding yeast counterparts. Unexpectedly, SpPrp18 and SpSlu7 were found by our lab to be required before catalysis and these proteins do not directly associate with each other. Genome-wide splicing studies in a missense slu7-2 mutant indicated widespread yet intron-specific splicing functions for SpSlu7 (Banerjee et al., 2013). Crucial functions were attributed to helix-5 and conserved region loop of SpPrp18 and in vivo splicing analysis in selected cellular transcripts in a missense mutant (V194R) also revealed intron-specific functions (Thesis, N Vijaykrishna). In this study, we have advanced our understanding of SpPrp18 functions by identifying its global substrates and correlating with its intron-specific roles. Through molecular and genetic approaches, we have probed its role in splicing/spliceosome assembly. We identified intronic features within substrates that increase the propensity for the requirement of SpSlu7 for efficient splicing. Further, using findings from the genome-wide alternative splicing patterns in SpSlu7 and SpPrp18 mutants, we have attempted to understand their role in splice-site choice and thus alternative splicing. Ia. Understanding global splicing functions and spliceosomal interactions of fission yeast splicing factor SpPrp18 Since SpPrp18 is an essential gene, our lab generated the strains (prp18-5int [V194R] and WTint), where the thiamine-repressible promoter allowed conditional expression of wild-type or mutant allele integrated at the heterologous leu1 locus. Splicing efficiency of certain cellular transcripts with differing intron characteristics was assessed by semi-quantitative RT-PCR studies and the data suggested intron-specific SpPrp18 roles (in collaboration with Vijaykrishna N). This prompted us to investigate the global splicing role for SpPrp18 for which we used splicing-sensitive microarrays having custom-designed probes to distinguish unspliced pre-mRNA and spliced mRNA for every individual pombe intron. RNA from prp18-5int (V194R) and WTint cells was used in these experiments. We derived a stringent dataset of 258 introns which were statistically significant and correlated in two biological replicate RNA samples, for various probes. Hierarchical clustering of this dataset showed that the depletion of wild-type SpPrp18 triggered a range of splicing phenotypes like (A) pre-mRNA accumulation with mRNA reduction (B) pre-mRNA accumulation (C) spliced mRNA reduction and (D) unchanged pre-mRNA and mRNA levels. Statistical analysis of cis motifs that may correlate with the substrate-specific SpPrp18 splicing functions was done, but the data showed a lack of a global discriminatory primary sequence feature. However, a subtle intron-specific role for Py(n) tracts located between 5’ss and BrP was deduced for SpPrp18. This lead was validated by examining the in vivo splicing efficiency of minitranscripts with wild-type or an altered Py tract length, carried out for a SpPrp18 dependent and an independent intron. To specifically address if SpPrp18 activity was required for second-step splicing we investigated, using primer extension analyses, for lariat intron-3’exon species, an intermediate formed after step 1. We observed that even in prp18-5int dbr1∆ double mutants (where lariat molecules are not degraded) the cells accumulate only unspliced pre-mRNA and not lariat intermediates, a signature of an early arrest prior to the first transesterification reaction. Strengthening these findings, positive genetic interactions were noted between prp18-5int and ts mutants in two factors (U2AF59 and SpPrp1) involved in precatalytic spliceosome assembly and activation. On the whole, our genome-wide studies indicate intron-specific pre-catalytic functions for SpPrp18 supported by genetic interactions with early acting splicing factors involved in spliceosomal assembly and activation. Ib. Identification of intronic features that determine substrate-specific splicing functions for SpSlu7 In vitro studies with ScSlu7 and hSlu7 show their influence in 3’ss selection when BrP to 3’ss distance is greater than 7 nts and 23 nts respectively; but the global substrates are not known in either species (Brys and Schwer, 1996; Chua and Reed, 1999b). Genome-wide analysis of the splicing efficiency changes in cells with the mis-sense spslu7+ mutant (slu7-2), previously carried out in our lab, revealed a spectrum of splicing defects (Banerjee et al., 2013). To further understand the intron context-specific roles for SpSlu7, we examined intronic cis features that may correlate with SpSlu7 dependence. Statistical analyses of the affected (422 introns) and unaffected categories (90 introns) revealed that intron length, BrP to 3’ss distance and AU content are multiple discriminatory cis features that govern SpSlu7 splicing functions. To assess the contribution of these intronic features we tested whether altering these cis elements changes a transcript’s dependency (or otherwise) on SpSlu7 by RT-PCR analyses. For these studies, we generated plasmid expressed mini-genes containing the respective wild-type intron or intron with altered BrP-3’ss distances. We used nab2+ I2 as a case of an intron spliced independent of SpSlu7 and rhb1+ I1 as a representative for SpSlu7 dependent intron. Experiments testing their in vivo splicing status proved that BrP-3’ss distance is a cis feature that dictates SpSlu7 splicing functions in a context-dependent manner. The intronic AU content particularly between the 5’ss and the BrP was assessed in minigene constructs where a chimeric intron was generated by swapping the low AU containing sequences in the 5’ss to BrP stretch of cdc2+ I2 with AU rich bpb1+ I1 5’ end sequences. The results reaffirmed that low intronic AU content particularly at the 5’ end co-relates with SpSlu7 dependency. Hence, we have deduced novel intronic elements, which perhaps in combination, create a contextual dependence for SpSlu7 to facilitate efficient splicing. II. Alternative splice-site selection in fission yeast and studies on the role of splicing factors SpSlu7 and SpPrp18 Budding yeast second-step splicing factors ScSlu7 and ScPrp18 mediate 3’ss choice in the single intron containing transcripts. Fission yeast genome encodes cis and trans factors that promote alternative splicing similar to higher eukaryotes. In this study, we have devised a data analysis pipeline to identify alternative splice events in multi-intronic transcripts of fission yeast. Further, we utilised this information to interrogate the global role for SpSlu7 and SpPrp18 in alternate splice site selection. We mapped the microarray probe sequences corresponding to all theoretically possible non-consecutive splice junctions of S. pombe transcripts onto two independent experimental next-generation (NGS) transcriptomes from wild-type samples and identified 104 exon skipping events with NGS reads more than 3 (Wilhelm et al., 2008; Rhind et al., 2011). We further generated a stringent list of ten exon skipping events having high sequence reads as well as raw intensity value in our microarray experiments with wild-type cells. Two representative events from this list, an abundant rps13+ exon 2 skipped alternative mRNA and less abundant ats1+ exon 3 skipped alternative mRNA were then taken up for experimental analyses by semi-quantitative RT-PCR assays. We confirmed these events and further noted that SpSlu7 and SpPrp18 were required for the constitutive splicing of ats1+ E2-I2-E3-I3-E4 cassette. On the other hand, SpSlu7, and not SpPrp18, exerted a subtle influence on the skipping of exon 3. In addition to exon 3 skipped mRNA, we detected an intron 3 retained ats1+ alternative mRNA (E2-E3-I3-E4) in wild-type cells. Assessment of this event in cells metabolically depleted of SpSlu7 and SpPrp18 showed a reduced abundance of this species in both instances. This suggests a role for functional SpSlu7 and SpPrp18 in retaining intron 3 in ats1+ transcripts in vivo. Among the ten microarray probes, custom-designed to detect specifically the mRNA isoforms arising from altered use of donor 5’ splice sites, we were able to detect in wild-type cells the utilisation of a downstream alternate 5’ss in intron 1 of D-Tyr-tRNA deacylase. Comparative assessment of this splicing event in prp18-5int and slu7-2 mutant cells revealed that SpPrp18 is preferentially required for the utilisation of its alternative 5’ss and such a role has not yet been attributed to its budding yeast and human homologs. On the other hand, SpSlu7 was required equally for utilisation of canonical and non-canonical 5’ss. Differential requirement for SpSlu7 for the utilisation of an upstream non-canonical 3’ss and the canonical 3’ss in DUF3074 intron 1, was noted. This role of SpSlu7 in 3’ss selection is similar to that known from in vitro studies of its budding yeast and human counterparts. Overall, we identified and experimentally validated novel alternate splice events in fission yeast and we infer an important role for SpSlu7 and SpPrp18 in both 5’ss and 3’ss selection.