No description available.

The spliceosome is a complex, dynamic ribonucleoprotein (RNP) complex that undergoes numerous conformational changes during its assembly, activation, catalysis and disassembly. Defects in spliceosome assembly are thought to trigger active dissociation of faulty splicing complexes. A yeast genetic screen was performed to identify components of the putative discard pathway. This study found that weak mutant alleles of SPP382 suppress defects in several proteins required for spliceosome activation (Prp38p, Prp8p and Prp19p) as well as substrate mutations (weak branch point mutants). This evolutionary conserved protein had been found in both yeast and mammalian splicing complexes. However, its role in splicing had not been elucidated. This study focused on understanding the cellular role of Spp382p in splicing and particularly in the discard pathway. Spp382p was found to be essential for normal splicing and for efficient intron turnover. Since Spp382p binds Prp43p and is required for intron release in vitro, spp382 mediated suppression of splicing factor mutations might reflect lowered Prp43p activity. In agreement with this, we find that prp43 mutants also act as suppressors. This leads us to propose a model in which defects in spliceosome assembly, like those caused by prp38-1, prompt Spp382p-mediated disassembly of the defective complex via Prp43p Bolstering this theory, we find that Spp382p is specifically recruited to defective complexes lacking the 5 exon cleavage intermediate and spp382 mutants suppress other splicing defects. I show by stringent proteomic and two-hybrid analyses that Spp382p interacts with Cwc23p, a DnaJ-like protein present in the spliceosome and co-purified the Prp43p-DExD/H-box protein. In this study, I also show that Cwc23p is itself essential for splicing and normal intron turnover. Enhanced expression of another protein, Sqs1p, structurally related to Spp382p and also found associated with Prp43p is inhibitory to both growth and splicing. Synthetic lethal and dosage suppression studies bolster a functional linkage between Spp382p, Cwc23p, Sqs1p and Prp43p and together, the data support the existence of a Spp382p -dependent spliceosome integrity (SPIN) complex acting to remove defective spliceosomes.

The U5 snRNP is a major component of the yeast spliceosome, being part of the U4/U6.U5 tri-snRNP, the precatalytic spliceosome and the catalytically activated spliceosome. The U5 snRNP includes, at its heart, the U5 snRNA which contains the invariant Loop 1 that functions in tethering and aligning exons during splicing. The major protein components of the U5 snRNP are the highly conserved Prp8p, the GTPase Snu114p and the helicase Brr2p. These proteins and the U5 snRNA are integral in forming the active site of the spliceosome and regulating the dynamic changes of the spliceosome. The first part of this study aimed to express and purify specific domains of Snu114p to define the structure and function of Snu114p. The N-terminal region of Snu114p was successfully expressed and purified from bacteria. Addition of the Snu114p N-terminal fragment to in vitro splicing assays resulted in a first step splicing defect, indicating a role for the N-terminus in pre-mRNA splicing. NMR studies revealed that the N-terminus of Snu114p exists as an unstructured protein domain. Mutagenesis indicated that the N-terminus of Snu114p is tolerant to mutation. A novel genetic interaction between amino acids in the N-terminus of Snu114p and the 3’ side of the U5 snRNA IL1 was identified. It is proposed here that the N-terminus of Snu114p functions to stabilise interactions of Snu114p with other proteins or snRNAs, possibly the U5 snRNA. Alternatively, the N-terminus of Snu114p may form intramolecular interactions with other regions of Snu114p to regulate Snu114p function in pre-mRNA splicing.Prp8p, Snu114p and Brr2p are known to form a stable complex but their interactions with the specific domains of the U5 snRNA are not known. The second part of this study aimed to investigate the association of Brr2p, Snu114p and Prp8p with the U5 snRNA. Mutants of the U5 snRNA were constructed in the conserved Loop 1 and the Internal Loop 1 (IL1). The influences of the U5 snRNA mutations on interactions of Prp8p, Snu114p or Brr2p with the snRNA were investigated. It was revealed that Loop 1 and both sides of IL1 of the U5 snRNA are important in association of Brr2p, Snu114p and Prp8p. Mutations in the 3’ side of IL1 drastically reduce association of Brr2p, Snu114p and Prp8p with the U5 snRNA, highlighting this region as a potential ‘protein docking’ site within the U5 snRNP. Differences seen in the associations of Brr2p, Snu114p and Prp8p with U5 snRNA mutations demonstrate that although there are intimate interactions between Brr2p, Snu114p and Prp8p, they do not associate with the U5 snRNA as a tri-protein complex. Genetic screening of BRR2 and U5 snRNA mutants reveals an interaction between the N-terminal half of Brr2p and the 3’ side of U5 snRNA IL1. This supports the proposed ‘protein docking’ site at the 3’ side of the U5 snRNA IL1.Data presented in this study increases our understanding of the regions in the U5 snRNA required for association of the essential U5 snRNP proteins, Brr2p, Snu114p and Prp8p, and goes some way to elucidating the organisation of essential proteins within the U5 snRNP.

No description available.

Pre-mRNA splicing and other RNA processing events occur co-transcriptionally. High resolution kinetic studies performed in our lab showed splicing-dependent RNA Pol II (RNA polymerase II) pausing near the 3’ splice site of a reporter gene. Pausing requires splicing, as mutations that block splicing lead to loss of pausing, and restoring splicing restores pausing. It was proposed that RNA Pol II pausing may occur at splicing-dependent transcriptional checkpoints. In this study, I aimed to search for splicing helicases that might couple splicing with transcription. The ts alleles prp5-1 and prp16-2 were found to cause transcription defects. These genes encode RNA helicases that were reported to act as fidelity factors during splicing. In vivo RNA labelling and RT-qPCR experiments performed with these temperature-sensitive mutants demonstrated reduced transcription coinciding with the splicing defect at restrictive temperature. Furthermore, RNA Pol II ChIP analysis showed polymerase accumulating over intron-containing genes in both mutants. ChIP analysis using antibodies specific to the phosphorylation status of the CTD (Carboxy Terminal Domain) of RNA Pol II, revealed that the apparently stalled polymerase is hyper-phosphorylated at serine 5. Intriguingly, prp8-R1753K, a ts allele of PRP8, a non-helicase splicing factor mutant also showed reduced nascent RNA synthesis but no RNA Pol II accumulation. To elucidate the reason for the observed RNA Pol II accumulation and to identify a possible splicing-dependent transcriptional checkpoint factor, prp5-1 was investigated further. RNA Pol II ChIP-Seq analysis verified that maximum enrichment genome-wide occurred on introns at restrictive conditions in prp5-1, supporting the earlier observation. Furthermore, the double mutant strain cus2Δprp5-1 abolished the RNA Pol II accumulation observed in prp5-1 at restrictive temperature and restored transcription. Recreating a stalled spliceosome in a U2 mutant strain also showed RNA Pol II accumulation in the presence of Cus2p, as observed in prp5-1. My observations suggest a link between transcription and monitoring of splicing and indicate that Cus2p, a U2 snRNP associated protein, could be a checkpoint factor in transcription prior to pre-spliceosome formation. I speculate that fidelity factors may impose transcriptional checkpoints at different stages of splicing.

No description available.

Alternative pre-mRNA splicing is often controlled by cell signals (1-3). Membrane depolarization/calcium (Ca2+) signaling controls alternative splicing of a group of genes in neurons and endocrine cells (4-9), with important implications in memory formation or secretion of hormones and neurotransmitters (10-15). However, the underlying molecular basis remains largely unknown. In rat GH3 pituitary cells, BK potassium channels control cellular electrical firing, which is critical for the release of growth hormone and prolactin. Inclusion of the STREX exon of the Slo1 gene encoding the channel α subunit is repressed by the Ca2+/calmodulin-dependent kinase IV (CaMKIV) upon depolarization (4). We isolated CaMKIV-responsive RNA elements (CaRREs) from a library of 13-nucleotide random sequences through in vivo selection in HEK293T cells. Most elements are CA-rich or A-rich, with the heterogeneous nuclear ribonucleoprotein (hnRNP) L as a binding factor. This is consistent with the finding that CA-rich elements and hnRNP L are targeted by CaMKIV in the regulation of splicing (16). In further efforts to directly link the kinase with hnRNP L, we showed that hnRNP L is essential for the full repression of STREX by depolarization and that a highly conserved CaMKIV target serine (Ser513) of L is required. Ser513 phosphorylation enhanced L binding to the STREX CaRRE1, leading to reduced binding of the constitutive factor U2AF65 to the 3’ splice site of STREX. Mutation of Ser513 abolished both activities. Therefore, hnRNP L mediates the repression of STREX by depolarization through modulation of a key step in spliceosomal assembly. We further identified hnRNP L, L-like (LL) and PTB as repressors of STREX and other depolarization-regulated exons with differential effects. Moreover, a full response of STREX to depolarization is mediated by combinations of hnRNP L and LL or PTB. Another depolarization-responsive exon, the exon 18 of the neuregulin 1 gene, is also controlled in a similar way, with the hnRNP L Ser513 required as well. This work provides the first direct link between the Ca2+ signaling and a specific serine of a regulatory splicing factor. Elucidation of the underlying molecular mechanisms would likely help us understand the fine-tuning of hormone secretion and memory formation.

Nuclear precursor of mature messenger RNA (pre-mRNA) splicing is one of the most highly regulated processes in eukaryotic organisms. In addition to its role in the removal of constitutive or alternative introns present in the pre-mRNA, splicing is also highly integrated into other layers of gene expression. This study investigates the potential role of the nuclear branchpoint binding protein (BBP) outside of the pre-mRNA splicing cycle. More specifically, we were interested in the biological relevance of its association with two cytoplasmic proteins Smy2 and Syh1. Smy2 and Syh1 belong to the GYF family of poly-proline binding proteins, and their roles in cell biology have not been well elucidated. Here we report that Smy2 and Syh1 act redundantly in: (i) limiting pre-mRNA accumulation when yeast cultures reach high cell density, potentially through promoting pre-mRNA decay in the cytoplasm; (ii) restricting Ty1 retrotransposition, apparently by limiting the Ty1 transcript abundance; (iii) limiting the accumulation of BBP-associated yet intronless TDA1 mRNA. With the presence of UACUAAC motif and BBP association as common features of these Smy2/Syh1 sensitive substrates, we tested if BBP interaction is required for Smy2/Syh1 function in RNA metabolism. Interestingly, we found that deletion of BBP C-terminal region (bbp∆C), which largely reduces or abolishes its association with Smy2, does not lead to similar phenotypes as observed in smy2∆ syh1∆ deletion mutant cells. In addition, mutagenesis of the TACTAAC BBP-binding site within the TDA1 coding region does not seem to affect TDA1 mRNA abundance or its sensitivity to the smy2∆ syh1∆ deletions. Therefore, we concluded that while the two BBP-binding proteins Smy2 and Syh1 impact the levels of certain cellular RNAs, this phenomenon is not strictly dependent upon BBP-Smy2 interaction and may be independent of BBP contribution. A model is proposed for Smy2 and Syh1 function in RNA metabolism based on our observations and interactions between these proteins with other factors implicated in RNA stability or translation.

Alternative pre-mRNA splicing is often controlled by cell signals (1-3). Membrane depolarization/calcium (Ca2+) signaling controls alternative splicing of a group of genes in neurons and endocrine cells (4-9), with important implications in memory formation or secretion of hormones and neurotransmitters (10-15). However, the underlying molecular basis remains largely unknown. In rat GH3 pituitary cells, BK potassium channels control cellular electrical firing, which is critical for the release of growth hormone and prolactin. Inclusion of the STREX exon of the Slo1 gene encoding the channel α subunit is repressed by the Ca2+/calmodulin-dependent kinase IV (CaMKIV) upon depolarization (4). We isolated CaMKIV-responsive RNA elements (CaRREs) from a library of 13-nucleotide random sequences through in vivo selection in HEK293T cells. Most elements are CA-rich or A-rich, with the heterogeneous nuclear ribonucleoprotein (hnRNP) L as a binding factor. This is consistent with the finding that CA-rich elements and hnRNP L are targeted by CaMKIV in the regulation of splicing (16). In further efforts to directly link the kinase with hnRNP L, we showed that hnRNP L is essential for the full repression of STREX by depolarization and that a highly conserved CaMKIV target serine (Ser513) of L is required. Ser513 phosphorylation enhanced L binding to the STREX CaRRE1, leading to reduced binding of the constitutive factor U2AF65 to the 3’ splice site of STREX. Mutation of Ser513 abolished both activities. Therefore, hnRNP L mediates the repression of STREX by depolarization through modulation of a key step in spliceosomal assembly. We further identified hnRNP L, L-like (LL) and PTB as repressors of STREX and other depolarization-regulated exons with differential effects. Moreover, a full response of STREX to depolarization is mediated by combinations of hnRNP L and LL or PTB. Another depolarization-responsive exon, the exon 18 of the neuregulin 1 gene, is also controlled in a similar way, with the hnRNP L Ser513 required as well. This work provides the first direct link between the Ca2+ signaling and a specific serine of a regulatory splicing factor. Elucidation of the underlying molecular mechanisms would likely help us understand the fine-tuning of hormone secretion and memory formation.

xiv, 59 p. : ill. (some col.) / Muscleblind-like 1 (MBNL1) is a splicing factor whose improper cellular localization is a central component of myotonic dystrophy (DM). In DM, the lack of properly localized MBNL1 leads to mis-splicing of many pre-mRNAs. The mechanism by which MBNL1 regulates it pre-mRNA targets is not well understood. In order to determine the mechanism by which MBNL1 regulates alternative splicing, a consensus RNA binding motif for Mbl (the <italic>Drosophila</italic> ortholog of MBNL1) and MBNL1 were determined using SELEX (Systematic Evolution of Ligands by Exponential Enrichment). These consensus motifs allowed for the identification of high affinity endogenous sites within pre-mRNAs that are regulated by MBNL1. <italic>In vitro</italic> binding studies showed that MBNL1 bound to RNAs that contained the consensus motif surrounded by pyrimidines. Some of these sites were identified upstream of exon 5 within the <italic>MBNL1</italic> pre-mRNA, and we have shown that MBNL1 auto-regulates the exclusion of exon 5 in HeLa cells. The region of the <italic>MBNL1</italic> gene that includes exon 5 and flanking intronic sequence is highly conserved in vertebrate genomes. The 3' end of intron 4 is non-canonical in that it contains an AAG 3' splice site and a predicted branchpoint that is 141 nucleotides from the 3' splice site. Using a mini-gene that includes exon 4, intron 4, exon 5, intron 5 and exon 6 of <italic>MBNL1</italic>, we show that MBNL1 regulates inclusion of exon 5. Mapping of the intron 4 branchpoint confirms that branching occurs primarily at the predicted distant branchpoint. Structure probing and footprinting reveal that the highly conserved region between the branchpoint and the 3' splice site is primarily unstructured, and MBNL1 binds within this region of the pre-mRNA, which we have termed the MBNL1 response element. Deletion of the response element eliminates MBNL1 splicing regulation and leads to complete inclusion of exon 5, which is consistent with the suppressive effect of MBNL1 on splicing. This dissertation includes previously published co-authored material as well as my recent co-authored material that has been submitted for publication. / Committee in charge: Kenneth Prehoda, Chair; J. Andrew Berglund, Advisor; Victoria J. de Rose, Member; Alice Barkan, Member; Karen Guillemin, Outside Member