How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing

James, Shelly-Ann; Turner, William H.; Schwer, Beate

doi:10.1017/s1355838202022033

Cited by 91 publications

(143 citation statements)

References 30 publications

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“…The interaction between Prp18 and Slu7 appears to be important for the proper function of both proteins+ Mutant Prp18s that fail to interact with Slu7 are invariably temperature sensitive+ These mutations can be suppressed to a similar extent by overexpression of Slu7 or by overexpression of the mutant prp18+ These (Horowitz & Abelson, 1993a;Brys & Schwer, 1996;Zhang & Schwer, 1997), but the possibility that Prp18 and Slu7 form a stable complex outside the spliceosome is not excluded by our results+ Our interpretation of the interactions of Prp18 and Slu7 is consistent with the recent work of James et al+ (2002), who studied the binding of Slu7 and Prp18 to the spliceosome in vitro+…”

Section: Interaction Of Prp18 With Slu7supporting

confidence: 85%

Mutational analysis identifies two separable roles of the Saccharomyces cerevisiae splicing factor Prp18

Bačíková

Horowitz

2002

RNA

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Prp18 functions in the second step of pre-mRNA splicing, joining the spliceosome just prior to the transesterification reaction that creates the mature mRNA. Prp18 interacts with Slu7, and the functions of the two proteins are intertwined. Using the X-ray structure of Prp18, we have designed mutants in Prp18 that imply that Prp18 has two distinct roles in splicing. Deletion mutations were used to delineate the surface of Prp18 that interacts with Slu7, and point mutations in Prp18 were used to define amino acids that contact Slu7. Experiments in which Slu7 and mutant Prp18 proteins were expressed at different levels support a model in which interaction between the proteins is needed for stable binding of both proteins to the spliceosome. Mutations in an evolutionarily conserved region show that it is critical for Prp18 function but is not involved in binding Slu7. Alleles with mutations in the conserved region are dominant negative, suggesting that the resulting mutant prp18 proteins make proper contacts with the spliceosome, but fail to carry out a Prp18-specific function. Prp18 thus appears to have two separable roles in splicing, one in stabilizing interaction of Slu7 with the spliceosome, and a second that requires the conserved loop.

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Section: Interaction Of Prp18 With Slu7supporting

confidence: 85%

Mutational analysis identifies two separable roles of the Saccharomyces cerevisiae splicing factor Prp18

Bačíková

Horowitz

2002

RNA

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“…The strong association between Prp18 and Slu7 aids their spliceosome assembly (55). Our prior studies suggested weak or no interactions between fission yeast SpPrp18 and SpSlu7 (22).…”

Section: Discussionmentioning

confidence: 83%

The Fission Yeast Pre-mRNA-processing Factor 18 (prp18+) Has Intron-specific Splicing Functions with Links to G1-S Cell Cycle Progression

Vijaykrishna¹,

Melangath²,

Kumar³

et al. 2016

Journal of Biological Chemistry

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Edited by Linda SpremulliThe fission yeast genome, which contains numerous short introns, is an apt model for studies on fungal splicing mechanisms and splicing by intron definition. Here we perform a domain analysis of the evolutionarily conserved Schizosaccharomyces pombe pre-mRNA-processing factor, SpPrp18. Our mutational and biophysical analyses of the C-terminal ␣-helical bundle reveal critical roles for the conserved region as well as helix five. We generate a novel conditional missense mutant, spprp18-5. To assess the role of SpPrp18, we performed global splicing analyses on cells depleted of prp18 ؉ and the conditional spprp18-5 mutant, which show widespread but intron-specific defects. In the absence of functional SpPrp18, primer extension analyses on a tfIId ؉ intron 1-containing minitranscript show accumulated pre-mRNA, whereas the lariat intron-exon 2 splicing intermediate was undetectable. These phenotypes also occurred in cells lacking both SpPrp18 and SpDbr1 (lariat debranching enzyme), a genetic background suitable for detection of lariat RNAs. These data indicate a major precatalytic splicing arrest that is corroborated by the genetic interaction between spprp18-5 and spprp2-1, a mutant in the early acting U2AF59 protein. Interestingly, SpPrp18 depletion caused cell cycle arrest before S phase. The compromised splicing of transcripts coding for G 1 -S regulators, such as Res2, a transcription factor, and Skp1, a regulated proteolysis factor, are shown. The cumulative effects of SpPrp18-dependent intron splicing partly explain the G 1 arrest upon the loss of SpPrp18. Our study using conditional depletion of spprp18 ؉ and the spprp18-5 mutant uncovers an intron-specific splicing function and early spliceosomal interactions and suggests links with cell cycle progression.Pre-mRNA splicing, a fundamental step in the processing of nascent eukaryotic RNA polymerase II transcripts, achieves precise excision of introns coupled with exon ligation to generate functional mRNAs. The spliceosome, which is composed of five U snRNPs 7 and Ͼ100 auxiliary proteins, assembles onto cis splicing signals, namely the 5Ј splice site (5Јss), the branch point nucleotide, the 3Ј splice site (3Јss), and polypyrimidine (Pyn) tracts. The spliceosomal catalytic core consists of the U2, U5, and U6 snRNPs and accessory proteins. This complex mediates the two trans-esterification reactions required for splicing to occur. First, the 5Јss is cleaved to yield the branched lariat intron-exon 2 and exon 1 intermediates, followed by the second reaction, where the 3Јss is cleaved, the exons are joined, and lariat intron is excised (1, 2).Genetic and biochemical analyses in budding yeast and biochemical studies with mammalian cell extracts have established a network of interactions among factors that act at the second step of splicing (i.e. Prp8, Prp16, Prp17, Prp18, Slu7, and Prp22) (3-8). PRP18, a non-essential budding yeast gene, encodes a U5 snRNP-associated factor. Prp18 has been analyzed extensively in budding yeast and human cell...

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“…After the first step of splicing, ATP hydrolysis by Prp16 leads to protection of the 3Ј splice site (7). We think that the ATP hydrolysis activates the spliceosome, permitting binding of Slu7, Prp18, and Prp22, and that this second-step spliceosome selects the 3Ј splice site and carries out the reaction (similar to the scheme of (9,10)). This model has a single kinetic step for identifying the 3Ј splice site and carrying out the reaction.…”

Section: Discussionmentioning

confidence: 94%

“…After the first transesterification, the RNA helicase Prp16 rearranges the spliceosome (7). Prp18, Slu7, and Prp22 join the spliceosome, which then rapidly ligates the exons (8)(9)(10). The U2, U5, and U6 snRNAs and the Prp8 protein, which are required for the first step of splicing, also participate directly in the second step (11,12).…”

mentioning

confidence: 99%

Exon sequences at the splice junctions affect splicing fidelity and alternative splicing

Crotti

Horowitz²

2009

Proc. Natl. Acad. Sci. U.S.A.

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Identification of splice sites is essential for the expression of most eukaryotic genes, allowing accurate splicing of pre-mRNAs. The splice sites are recognized by the splicing machinery based on sequences within the pre-mRNA. Here, we show that the exon sequences at the splice junctions play a significant, previously unrecognized role in the selection of 3 splice sites during the second step of splicing. The influence of the exon sequences was enhanced by the Prp18 mutant Prp18⌬CR, and the strength of an exon sequence in Prp18⌬CR splicing predicted its effect in wild-type splicing. Analysis of the kinetics of splicing in vitro demonstrated that 3 splice sites were chosen competitively during the second step, likely at the same time as exon ligation. In wild-type yeast, splice site selection for two genes studied was altered by point mutations in their exon bases, affecting splicing fidelity and alternative splicing. Finally, we note that the degeneracy of the genetic code allows competing 3 splice sites to be eliminated from coding regions, and we suggest that the evolution of the splicing signals and the genetic code are connected.pre-mRNA splicing ͉ Prp18 ͉ splice site selection I dentification of splice sites is necessary both for ensuring the fidelity of pre-mRNA splicing and for the regulation of alternative splicing. Mutations that impair the recognition of the splice sites are responsible for a significant fraction of genetic diseases (1). Splice sites are defined by sequence elements in the pre-mRNA that are recognized by many splicing factors (2). The 3Ј splice site is usually the first AG downstream of the branchpoint and is immediately preceded by a pyrimidine or an A (3, 4). A polypyrimidine tract is often present upstream of the splice site. The influence of each element varies and not all are required (5). Recognition of elements in the 3Ј splice site region is required early in splicing in metazoans but not in yeast (6).Identification of the 3Ј splice site for exon ligation appears to occur in the second step, and these events are well studied in yeast. After the first transesterification, the RNA helicase Prp16 rearranges the spliceosome (7). Prp18, Slu7, and Prp22 join the spliceosome, which then rapidly ligates the exons (8-10). The U2, U5, and U6 snRNAs and the Prp8 protein, which are required for the first step of splicing, also participate directly in the second step (11,12).The 3Ј splice site is identified by various mechanisms. The distance between the branch point and the 3Ј splice site is important, with distant sites dependent on their polypyrimidine tracts (13,14). The G's at the 5Ј and 3Ј ends of the intron interact (13, 15), and base Ϫ3 at the 3Ј site interacts with base ϩ3 at the 5Ј site and the U6 snRNA (16). Prp8 is involved in recognition of both the polypyrimidine tract and the AG dinucleotide at the 3Ј splice site (12, 17). Mutation of Slu7 impairs the use of distant sites (18,19). Analyses of in vivo splicing suggest that the 3Ј splice site recognition events occur during th...

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How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing

Cited by 91 publications

References 30 publications

Mutational analysis identifies two separable roles of the Saccharomyces cerevisiae splicing factor Prp18

Mutational analysis identifies two separable roles of the Saccharomyces cerevisiae splicing factor Prp18

The Fission Yeast Pre-mRNA-processing Factor 18 (prp18+) Has Intron-specific Splicing Functions with Links to G1-S Cell Cycle Progression

Exon sequences at the splice junctions affect splicing fidelity and alternative splicing

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