The RNA binding protein Cwc2 interacts directly with the U6 snRNA to link the nineteen complex to the spliceosome during pre-mRNA splicing

Intron removal during pre-messenger RNA (pre-mRNA) splicing involves arrangement of snRNAs into conformations that promote the two catalytic steps. The Prp19 complex [nineteen complex (NTC)] can specify U5 and U6 snRNA interactions with pre-mRNA during spliceosome activation. A candidate for linking the NTC to the snRNAs is the NTC protein Cwc2, which contains motifs known to bind RNA, a zinc finger and RNA recognition motif (RRM). In yeast cells mutation of either the zinc finger or RRM destabilize Cwc2 and are lethal. Yeast cells depleted of Cwc2 accumulate pre-mRNA and display reduced levels of U1, U4, U5 and U6 snRNAs. Cwc2 depletion also reduces U4/U6 snRNA complex levels, as found with depletion of other NTC proteins, but without increase in free U4. Purified Cwc2 displays general RNA binding properties and can bind both snRNAs and pre-mRNA in vitro. A Cwc2 RRM fragment alone can bind RNA but with reduced efficiency. Under splicing conditions Cwc2 can associate with U2, U5 and U6 snRNAs, but can only be crosslinked directly to the U6 snRNA. Cwc2 associates with U6 both before and after the first step of splicing. We propose that Cwc2 links the NTC to the spliceosome during pre-mRNA splicing through the U6 snRNA.     

The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis

Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 degrees C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Delta extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) but it is not a core component of any one snRNP. Prp17 association with in-vitro-assembled spliceosome complexes on actin pre-mRNAs was also investigated. Although the U5 snRNP proteins Prp8 and Snu114 are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step; however, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus Prp17 joins the spliceosome prior to both catalytic reactions. Our results indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17-spliceosome association analyses provide a biochemical basis for the delayed first step in prp17Delta and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.     

Intron Retention in the Alternatively Spliced Region of RON Results from Weak 3’ Splice Site Recognition

The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3’ splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5’ of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo.     

Stable tri-snRNP integration is accompanied by a major structural rearrangement of the spliceosome that is dependent on Prp8 interaction with the 5′ splice site

Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5′ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5′ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5′ss sequence, most importantly between Prp8 and nucleotides at the exon–intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.     

DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle

The DExD/H-box Prp5 protein (Prp5p) is an essential, RNA-dependent ATPase required for pre-spliceosome formation during nuclear pre-mRNA splicing. In order to understand how this protein functions, we used in vitro, biochemical assays to examine its association with the spliceosome from Saccharomyces cerevisiae. GST-Prp5p in splicing assays pulls down radiolabeled pre-mRNA as well as splicing intermediates and lariat product, but reduced amounts of spliced mRNA. It cosediments with active spliceosomes isolated by glycerol gradient centrifugation. In ATP-depleted extracts, GST-Prp5p associates with pre-mRNA even in the absence of spliceosomal snRNAs. Maximal selection in either the presence or absence of ATP requires a pre-mRNA with a functional intron. Prp5p is present in the commitment complex and functions in subsequent pre-spliceosome formation. Reduced Prp5p levels decrease levels of commitment, pre-spliceosomal and spliceosomal complexes. Thus Prp5p is most likely an integral component of the spliceosome, being among the first splicing factors associating with pre-mRNA and remaining until spliceosome disassembly. The results suggest a model in which Prp5p recruits the U2 snRNP to pre-mRNA in the commitment complex and then hydrolyzes ATP to promote stable association of U2 in the pre-spliceosome. They also suggest that Prp5p could have multiple ATP-independent and ATP-dependent functions at several stages of the splicing cycle.     

Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes

Excision of introns from pre-mRNAs is mediated by the spliceosome, a multi-megadalton complex consisting of U1, U2, U4/U6, and U5 snRNPs plus scores of associated proteins. Spliceosome assembly and disassembly are highly dynamic processes involving multiple stable intermediates. In this study, we utilized a split TAP-tag approach for large-scale purification of an abundant endogenous U2·U5·U6 complex from Schizosaccharomyces pombe. RNAseq revealed this complex to largely contain excised introns, indicating that it is primarily ILS (intron lariat spliceosome) complexes. These endogenous ILS complexes are remarkably resistant to both high-salt and nuclease digestion. Mass spectrometry analysis identified 68, 45, and 43 proteins in low-salt-, high-salt-, and micrococcal nuclease-treated preps, respectively. The protein content of a S. pombe ILS complex strongly resembles that previously reported for human spliced product (P) and Saccharomyces cerevisiae ILS complexes assembled on single pre-mRNAs in vitro. However, the ATP-dependent RNA helicase Brr2 was either substoichiometric in low-salt preps or completely absent from high-salt and MNase preps. Because Brr2 facilitates spliceosome disassembly, its relative absence may explain why the ILS complex accumulates logarithmically growing cultures and the inability of S. pombe extracts to support in vitro splicing.     

A distinct complex of PRP19-related and trypanosomatid-specific proteins is required for pre-mRNA splicing in trypanosomes

The pre-mRNA splicing factor PRP19 is recruited into the spliceosome after forming the PRP19/CDC5L complex in humans and the Nineteen complex in yeast. Additionally, ‘PRP19-related’ proteins enter the spliceosome individually or in pre-assemblies that differ in these systems. The protistan family Trypanosomatidae, which harbors parasites such as Trypanosoma brucei, diverged early during evolution from opisthokonts. While introns are rare in these organisms, spliced leader trans splicing is an obligatory step in mRNA maturation. So far, ∼70 proteins have been identified as homologs of human and yeast splicing factors. Moreover, few proteins of unknown function have recurrently co-purified with splicing proteins. Here we silenced the gene of one of these proteins, termed PRC5, and found it to be essential for cell viability and pre-mRNA splicing. Purification of PRC5 combined with sucrose gradient sedimentation revealed a complex of PRC5 with a second trypanosomatid-specific protein, PRC3, and PRP19-related proteins SYF1, SYF3 and ISY1, which we named PRP19-related complex (PRC). Importantly, PRC and the previously described PRP19 complex are distinct from each other because PRC, unlike PRP19, co-precipitates U4 snRNA, which indicates that PRC enters the spliceosome prior to PRP19 and uncovers a unique pre-organization of these proteins in trypanosomes.     

Yeast ntr1/spp382 mediates prp43 function in postspliceosomes

The Ntr1 and Ntr2 proteins of Saccharomyces cerevisiae have been reported to interact with proteins involved in pre-mRNA splicing, but their roles in the splicing process are unknown. We show here that they associate with a postsplicing complex containing the excised intron and the spliceosomal U2, U5, and U6 snRNAs, supporting a link with a late stage in the pre-mRNA splicing process. Extract from cells that had been metabolically depleted of Ntr1 has low splicing activity and accumulates the excised intron. Also, the level of U4/U6 di-snRNP is increased but those of the free U5 and U6 snRNPs are decreased in Ntr1-depleted extract, and increased levels of U2 and decreased levels of U4 are found associated with the U5 snRNP protein Prp8. These results suggest a requirement for Ntr1 for turnover of the excised intron complex and recycling of snRNPs. Ntr1 interacts directly or indirectly with the intron release factor Prp43 and is required for its association with the excised intron. We propose that Ntr1 promotes release of excised introns from splicing complexes by acting as a spliceosome receptor or RNA-targeting factor for Prp43, possibly assisted by the Ntr2 protein.     

Arrested yeast splicing complexes indicate stepwise snRNP recruitment during in vivo spliceosome assembly

Pre-mRNA splicing is catalyzed by the spliceosome, a macromolecular machine dedicated to intron removal and exon ligation. Despite an abundance of in vitro information and a small number of in vivo studies, the pathway of yeast (Saccharomyces cerevisiae) in vivo spliceosome assembly remains uncertain. To address this situation, we combined in vivo depletions of U1, U2, or U5 snRNAs with chromatin immunoprecipitation (ChIP) analysis of other splicing snRNPs along an intron-containing gene. The data indicate that snRNP recruitment to nascent pre-mRNA predominantly proceeds via the canonical three-step assembly pathway: first U1, then U2, and finally the U4/U6*U5 tri-snRNP. Tandem affinity purification (TAP) using a U2 snRNP-tagged protein allowed the characterization of in vivo assembled higher-order splicing complexes. Consistent with an independent snRNP assembly pathway, we observed high levels of U1-U2 prespliceosomes under U5-depletion conditions, and we observed significant levels of a U2/U5/U6/Prp19-complex mature splicing complex under wild-type conditions. These complexes have implications for the steady-state distribution of snRNPs within nuclei and also reinforce the stepwise recruitment of U1, U2, and the tri-snRNP during in vivo spliceosome assembly.     

Dynamic Contacts of U2, RES,Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes

Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B^act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B^act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25’s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome''s protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.     

A cyclophilin functions in pre-mRNA splicing

We report that the cyclophilin USA-CyP is part of distinct complexes with two spliceosomal proteins and is involved in both steps of pre-mRNA splicing. The splicing factors hPrp18 and hPrp4 have a short region of homology that defines a high affinity binding site for USA-CyP in each protein. USA-CyP forms separate, stable complexes with hPrp18 and hPrp4 in which the active site of the cyclophilin is exposed. The cyclophilin inhibitor cyclosporin A slows pre-mRNA splicing in vitro, and we show that its inhibition of the second step of splicing is caused by blocking the action of USA-CyP within its complex with hPrp18. Cyclosporin A also slows splicing in vivo, and we show that this slowing results specifically from inhibition of USA-CyP. Our results lead to a model in which USA-CyP is carried into the spliceosome in complexes with hPrp4 and hPrp18, and USA-CyP acts during splicing within these complexes. These results provide an example of the function of a cyclophilin in a complex process and provide insight into the mechanisms of action of cyclophilins.     

Structural basis for the dual U4 and U4atac snRNA-binding specificity of spliceosomal protein hPrp31

Human proteins 15.5K and hPrp31 are components of the major spliceosomal U4 snRNP and of the minor spliceosomal U4atac snRNP. The two proteins bind to related 5'-stem loops (5'SLs) of the U4 and U4atac snRNAs in a strictly sequential fashion. The primary binding 15.5K protein binds at K-turns that exhibit identical sequences in the two snRNAs. However, RNA sequences contacted by the secondary binding hPrp31 differ in U4 and U4atac snRNAs, and the mechanism by which hPrp31 achieves its dual specificity is presently unknown. We show by crystal structure analysis that the capping pentaloops of the U4 and U4atac 5'SLs adopt different structures in the ternary hPrp31-15.5K-snRNA complexes. In U4atac snRNA, a noncanonical base pair forms across the pentaloop, based on which the RNA establishes more intimate interactions with hPrp31 compared with U4 snRNA. Stacking of hPrp31-His270 on the noncanonical base pair at the base of the U4atac pentaloop recapitulates intramolecular stabilizing principles known from the UUCG and GNRA families of RNA tetraloops. Rational mutagenesis corroborated the importance of the noncanonical base pair and the U4atac-specific hPrp31-RNA interactions for complex stability. The more extensive hPrp31-U4atac snRNA interactions are in line with a higher stability of the U4atac compared with the U4-based ternary complex seen in gel-shift assays, which may explain how U4atac snRNA can compete with the more abundant U4 snRNA for the same protein partners in vivo.     

Recognition of the 5' splice site by the spliceosome.

The splicing of nuclear pre-mRNAs is catalyzed by a large, multicomponent ribonucleoprotein complex termed the spliceosome. Elucidation of the molecular mechanism of splicing identified small nuclear RNAs (snRNAs) as important components of the spliceosome, which, by analogy to the self-splicing group II introns, are implicated in formation of the catalytic center. In particular, the 5' splice site (5'SS) and the branch site, which represent the two substrates for the first step of splicing, are first recognized by U1 and U2 snRNPs, respectively. This initial recognition of splice sites is responsible for the global definition of exons and introns, and represents the primary target for regulation of splicing. Subsequently, pairing interaction between the 5'SS and U1 snRNA is disrupted and replaced by a new interaction of the 5'SS with U6 snRNA. The 5'SS signal contains an invariant GU dinucleotide present at the 5' end of nearly all known introns, however, the mechanism by which the spliceosome recognizes this element is not known. We have identified and characterized a specific UV light-induced crosslink formed between the 5'SS RNA and hPrp8, a protein component of U5 snRNP in the spliceosome that is likely to reflect a specific recognition of the GU dinucleotide for splicing. Because recognition of the 5'SS must be linked to formation of the catalytic site, the identification of a specific and direct interaction between the 5'SS and Prp8 has significant implications for the role of this protein in the mechanism of mRNA splicing.     

A computational scan for U12-dependent introns in the human genome sequence

U12-dependent introns are found in small numbers in most eukaryotic genomes, but their scarcity makes accurate characterisation of their properties challenging. A computational search for U12-dependent introns was performed using the draft version of the human genome sequence. Human expressed sequences confirmed 404 U12-dependent introns within the human genome, a 6-fold increase over the total number of non-redundant U12-dependent introns previously identified in all genomes. Although most of these introns had AT-AC or GT-AG terminal dinucleotides, small numbers of introns with a surprising diversity of termini were found, suggesting that many of the non-canonical introns found in the human genome may be variants of U12-dependent introns and, thus, spliced by the minor spliceosome. Comparisons with U2-dependent introns revealed that the U12-dependent intron set lacks the ‘short intron’ peak characteristic of U2-dependent introns. Analysis of this U12-dependent intron set confirmed reports of a biased distribution of U12-dependent introns in the genome and allowed the identification of several alternative splicing events as well as a surprising number of apparent splicing errors. This new larger reference set of U12-dependent introns will serve as a resource for future studies of both the properties and evolution of the U12 spliceosome.