Prp16p, Slu7p, and Prp8p interact with the 3' splice site in two distinct stages during the second catalytic step of pre-mRNA splicing.

For the second catalytic step of pre-mRNA splicing to occur, a 3' splice site must be selected and juxtaposed with the 5' exon. Four proteins, Prp16p, Slu7p, Prp17p, Prp18p, and an integral spliceosomal protein, Prp8p, are known to be required for the second catalytic step. prp8-101, an allele of PRP8 defective in 3' splice site recognition, exhibits specific genetic interactions with mutant alleles of the other second step splicing factors. The prp8-101 mutation also results in decreased crosslinking of Prp8p to the 3' splice site. To determine the role of the step-two-specific proteins in 3' splice site recognition and in binding of Prp8p to the 3' splice site, we performed crosslinking studies in mutant and immunodepleted extracts. Our results suggest an ordered pathway in which, after the first catalytic step, Prp16p crosslinks strongly to the 3' splice site and Prp8p and Slu7p crosslink weakly. ATP hydrolysis by Prp16p affects a conformational change that reduces the crosslinking of Prp16p with the 3' splice site and allows stronger crosslinking of Prp8p and Slu7p. Thus, the 3' splice site appears to be recognized in two stages during the second step of splicing. Strong 3' splice site crosslinking of Prp8p and Slu7p also requires the functions of Prp17p and Prp18p. Therefore, Prp8p and Slu7p interact with the 3' splice site at the latest stage of splicing prior to the second catalytic step that can currently be defined, and may be at the active site.     

Mutagenesis of the Yeast Gene Prp8 Reveals Domains Governing the Specificity and Fidelity of 3' Splice Site Selection

PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and later steps of pre-mRNA splicing. We recently identified a novel allele, prp8-101, that specifically impairs recognition of the uridine tract that precedes most yeast 3'' slice sites. We carried out extensive mutagenesis of the gene and selected for new alleles that confer a phenotype similar to that of prp8-101. The strongest alleles cause changes in one of two amino acids in the C-terminal portion of the protein. We also identified a second class of PRP8 mutant that affects the fidelity of 3'' splice site utilization. These alleles suppress point mutations in the PyAG motif at the 3'' splice site and do not alter uridine tract recognition. The strongest of these alleles map to a region directly upstream of the prp8-101-like mutations. These new PRP8 alleles define two separable functions of Prp8p, required for specificity of 3'' splice site selection and fidelity of 3'' splice site utilization, respectively. Taken together with other recent biochemical and genetic data, our results suggest that Prp8p plays a functional role at the active site of the spliceosome during the second catalytic step of splicing.     

Genetic Studies of the Prp17 Gene of Saccharomyces Cerevisiae: A Domain Essential for Function Maps to a Nonconserved Region of the Protein

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the β transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the β transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is in the N terminal region of the protein.     

Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing.

Pre-mRNA splicing takes place in two catalytic steps. The second step is poorly understood, especially in mammals. In yeast, the splicing factors, Prps 16, 17, 18 and Slu7 function exclusively in step II. Here we report the isolation of cDNAs encoding human Prps 16 and 17 which are 41 and 36% identical to their yeast counterparts. The Prp16 gene is essential in yeast, and we show that a chimeric yeast-human Prp16 protein rescues a yeast Prp16 knockout strain. Immunodepletion of hPrp16 from splicing extracts specifically blocks step II, and the activity can be fully restored with recombinant hPrp16. Moreover, both hPrps 16 and 17 associate with the spliceosome late in the splicing pathway. Mutations at the 3' splice site that specifically block step II do not affect the association of hPrps 16 and 17 with the spliceosome, indicating that these factors may function at a stage of step II prior to recognition of the 3' splice site. Recently, the human homologs of Prp18 and Slu7 were identified. The observation that humans contain homologs of all four known step II proteins in yeast indicates that the mechanism for catalytic step II is highly conserved.     

The yeast PRP8 protein interacts directly with pre-mRNA.

The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.     

Evidence for a role of Sky1p-mediated phosphorylation in 3' splice site recognition involving both Prp8 and Prp17/Slu4

The SRPK family of kinases is specific for RS domain-containing splicing factors and known to play a critical role in protein-protein interaction and intracellular distribution of their substrates in both yeast and mammalian cells. However, the function of these kinases in pre-mRNA splicing remains unclear. Here we report that SKY1, a SRPK family member in Saccharomyces cerevisiae, genetically interacts with PRP8 and PRP17/SLU4, both of which are involved in splice site selection during pre-mRNA splicing. Prp8 is essential for splicing and is known to interact with both 5' and 3' splice sites in the spliceosomal catalytic center, whereas Prp17/Slu4 is nonessential and is required only for efficient recognition of the 3' splice site. Interestingly, deletion of SKY1 was synthetically lethal with all prp17 mutants tested, but only with specific prp8 alleles in a domain implicated in governing fidelity of 3'AG recognition. Indeed, deletion of SKY1 specifically suppressed 3'AG mutations in ACT1-CUP1 splicing reporters. These results suggest for the first time that 3' AG recognition may be subject to phosphorylation regulation by Sky1p during pre-mRNA splicing.     

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.     

Extensive interactions of PRP8 protein with the 5'' and 3'' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP.     

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing.

PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase T1 treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branch-point-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.     

Suppression of multiple substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants

Conformational change within the spliceosome is required between the first catalytic step of pre-mRNA splicing, when the branch site (BS) attacks the 5' splice site, and the second step, when the 5' exon attacks the 3' splice site, yielding mRNA and lariat-intron products. A genetic screen for suppressors of BS A-to-G mutants, which stall between the two steps, identified Prp8, the highly conserved spliceosomal factor. prp8 suppressors facilitate the second step for multiple intron mutants and interact functionally with first step suppressors, alleles of PRP16 and U6 snRNA. Genetic interactions among prp8, prp16, and U6 alleles suggest that these factors control a common stage in first-to-second step transition. We propose that mutant substrates are utilized by alteration of the equilibrium between first/second step conformations, resembling tRNA miscoding caused by altered equilibrium between open/closed ribosomal conformations. This mechanistic commonality suggests that alteration of rearrangements represents an evolutionarily convenient way of modulating substrate selectivity.     

A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis.

PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.     

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.     

The Saccharomyces cerevisiae gene CDC40/PRP17 controls cell cycle progression through splicing of the ANC1 gene

The timing of events in the cell cycle is of crucial importance, as any error can lead to cell death or cancerous growth. This accurate timing is accomplished through the activation of specific CDC genes. Mutations in the CDC40/PRP17 gene cause cell cycle arrest at the G₂/M stage. It was previously found that the CDC40 gene encodes a pre-mRNA splicing factor, which participates in the second step of the splicing reaction. In this paper we dissect the mechanism by which pre-mRNA splicing affects cell cycle progression. We identify ANC1 as the target of CDC40 regulation. Deletion of the ANC1 intron relieves the cell cycle arrest and temperature sensitivity of cdc40 mutants. Furthermore, we identify, through point mutation analysis, specific residues in the ANC1 intron that are important for its splicing dependency on Cdc40p. Our results demonstrate a novel mechanism of cell cycle regulation that relies on the differential splicing of a subset of introns by specific splicing factors.     

Sequence requirements for splicing by the Cne PRP8 intein

The dependence of protein splicing on conserved residues of the Cne PRP8 intein was assessed by alanine scanning mutagenesis in a foreign protein context. Corroboration was obtained for the involvement of residues at the splice junctions and of the conserved threonine and histidine of motif B. Five additional residues were identified as absolutely required for splicing. Variant W151A displayed premature C-terminal cleavage, not seen with other Cne PRP8 mutants. We propose a model whereby W151 acts to prevent premature C-terminal cleavage, favoring complete splicing as opposed to two disjointed cleavage events.     

Mutations at the 3' splice site can be suppressed by compensatory base changes in U1 snRNA in fission yeast.

U1 snRNA is an essential splicing factor known to base pair with 5' splice sites of premessenger RNAs. We demonstrate that pairing between the universally conserved CU just downstream from the 5' junction interaction region and the 3' splice site AG contributes to efficient splicing of Schizosaccharomyces pombe introns that typify the AG-dependent class described in mammals. Strains carrying mutations in the 3' AG of an artificial intron accumulate linear precursor, indicative of a first step block. Lariat formation is partially restored in these mutants by compensatory changes in nucleotides C7 and U8 of U1 snRNA. Consistent with a general role in fission yeast splicing, mutations at C7 are lethal, while U8 mutants are growth impaired and accumulate linear, unspliced precursor to U6 snRNA. U1 RNA-mediated recognition of the 3' splice site may have origins in analogous intramolecular interactions in an ancestral self-splicing RNA.     

Mutations in the U5 snRNA result in altered splicing of subsets of pre-mRNAs and reduced stability of Prp8

The U5 snRNA loop 1 aligns the 5′ and 3′ exons for ligation during the second step of pre-mRNA splicing. U5 is intimately associated with Prp8, which mediates pre-mRNA repositioning within the catalytic core of the spliceosome and interacts directly with U5 loop 1. The genome-wide effect of three U5 loop 1 mutants has been assessed by microarray analysis. These mutants exhibited impaired and improved splicing of subsets of pre-mRNAs compared to wild-type U5. Analysis of pre-mRNAs that accumulate revealed a change in base prevalence at specific positions near the splice sites. Analysis of processed pre-mRNAs exhibiting mRNA accumulation revealed a bias in base prevalence at one position within the 5′ exon. While U5 loop 1 can interact with some of these positions the base bias is not directly related to sequence changes in loop 1. All positions that display a bias in base prevalence are at or next to positions known to interact with Prp8. Analysis of Prp8 in the presence of the three U5 loop 1 mutants revealed that the most severe mutant displayed reduced Prp8 stability. Depletion of U5 snRNA in vivo also resulted in reduced Prp8 stability. Our data suggest that certain mutations in U5 loop 1 perturb the stability of Prp8 and may affect interactions of Prp8 with a subset of pre-mRNAs influencing their splicing. Therefore, the integrity of U5 is important for the stability of Prp8 during splicing and provides one possible explanation for why U5 loop 1 and Prp8 are so highly conserved.     

Prp43 is an essential RNA-dependent ATPase required for release of lariat-intron from the spliceosome

The essential Saccharomyces cerevisiae PRP43 gene encodes a 767-amino acid protein of the DEXH-box family. Prp43 has been implicated in spliceosome disassembly (Arenas, J. E., and Abelson, J. N. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11798-11802). Here we show that purified recombinant Prp43 is an RNA-dependent ATPase. Alanine mutations at conserved residues within motifs I ((119)GSGKT(123)), II ((215)DEAH(218)) and VI ((423)QRAGRAGR(430)) that diminished ATPase activity in vitro were lethal in vivo, indicating that ATP hydrolysis is necessary for the biological function of Prp43. Overexpression of lethal, ATPase-defective mutants in a wild-type strain resulted in dominant-negative growth inhibition. The ATPase-defective mutant T123A interfered in trans with the in vitro splicing function of wild-type Prp43. T123A did not affect the chemical steps of splicing or the release of mature mRNA from the spliceosome, but it blocked the release of the excised lariat-intron from the spliceosome. We show that the lariat-intron is not accessible to debranching by purified Dbr1 when it is held in the T123A-arrested splicing complex. Our results define a new ATP-dependent step of splicing that is catalyzed by Prp43.     

Functional interactions between Prp8, Prp18, Slu7, and U5 snRNA during the second step of pre-mRNA splicing

After the second transesterification step of pre-mRNA splicing, the Prp22 helicase catalyzes release of spliced mRNA by disrupting contacts in the spliceosome that likely involve Prp8. Mutations at Arg1753 in Prp8, which suppress helicase-defective prp22 mutants, elicit temperature-sensitive growth phenotypes, indicating that interactions in the spliceosome involving Prp8-R1753 might be broken prematurely at 37 degrees C. Here we report that mutations in loop I of the U5 snRNA or in Prp18 can suppress the temperature-sensitive prp8-R1753 mutants. The same gain-of-function PRP18 alleles can also alleviate the growth phenotypes of multiple slu7-ts mutants, indicating a functional link between Prp8 and the second step splicing factors Prp18 and Slu7. These findings, together with the demonstration that changes at Arg1753 in Prp8 impair step 2 of pre-mRNA splicing in vitro, are consistent with a model in which (1) Arg1753 plays a role in stabilizing U5/exon interactions prior to exon joining and (2) these contacts persist until they are broken by the helicase Prp22.