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

Dagher, Sue F.; Fu, Xiang‐Dong

doi:10.1017/s1355838201016077

Cited by 29 publications

(29 citation statements)

References 72 publications

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“…Prp8 is a U5 RNA-associated protein that contacts both the 5Ј and 3Ј splice sites of the intron (12)(13)(14)(15) as well as U6 RNA (16). Prp8 has been implicated in regulation of the second transesterification reaction (17)(18)(19)(20); our results suggest that Prp8 also functions in the first catalytic step of splicing. Although Prp8 is two-thirds identical between yeast and humans (21), it contains no recognizable structural motifs.…”

mentioning

confidence: 84%

Distinct domains of splicing factor Prp8 mediate different aspects of spliceosome activation

Kuhn

Reichl

Brow

2002

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

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Prp8 is the largest and most highly conserved protein in the spliceosome yet its mechanism of function is poorly understood. Our previous studies implicate Prp8 in control of spliceosome activation for the first catalytic step of splicing, because substitutions in five distinct regions (a-e) of Prp8 suppress a cold-sensitive block to activation caused by a mutation in U4 RNA. Catalytic activation of the spliceosome is thought to require unwinding of the U1 RNA͞5 splice site and U4͞U6 RNA helices by the Prp28 and Prp44͞Brr2 DExD͞H-box helicases, respectively. Here we show that mutations in regions a, d, and e of Prp8 exhibit allele-specific genetic interactions with mutations in Prp28, Prp44͞Brr2, and U6 RNA, respectively. These results indicate that Prp8 coordinates multiple processes in spliceosome activation and enable an initial correlation of Prp8 structure and function. Furthermore, additional genetic interactions with U4-cs1 support a two-state model for this RNA conformational switch and implicate another splicing factor, Prp31, in Prp8-mediated spliceosome activation.pre-mRNA splicing ͉ small nuclear RNA ͉ retinitis pigmentosa ͉ RNA helicases ͉ Prp31 T he removal of introns from nuclear pre-mRNA is catalyzed by a macromolecular complex called the spliceosome, which consists of five RNAs (U1, U2, U4, U5, and U6) and more than 70 proteins. Pre-mRNA splicing is thought to be RNA-catalyzed and involves two transesterification reactions (1). The accuracy and efficiency of splicing is achieved by means of a high degree of regulation, imposed by a complex series of protein-protein, protein-RNA, and RNA-RNA interactions. The spliceosome either assembles on the intron by way of stepwise recruitment of the constituent small nuclear ribonucleoproteins, each containing one RNA and several proteins (2), or a fully assembled spliceosome binds the intron as a unit (3). After the complete spliceosome has engaged an intron, it must undergo extensive conformational rearrangements to become catalytically active. Early steps in catalytic activation include unwinding of the U1 RNA͞5Ј splice site helix and unwinding of the U4͞U6 helices (Fig. 1). These unwinding events are thought to be catalyzed by two DExD͞H-box RNA helicases, Prp28 (4, 5) and Prp44͞Brr2 (6-8), respectively.We identified a cold-sensitive mutation in U4 RNA, called U4-cs1, which blocks catalytic activation at low temperature (9, 10). U4-cs1 has a triple nucleotide substitution that extends the U4͞U6 base-pairing, thereby masking the ''ACAGA'' sequence in U6 RNA that base pairs with the 5Ј splice site when U1 RNA is displaced (Fig. 1). We proposed that in the absence of proper U6 RNA͞5Ј splice site pairing, U4͞U6 RNA unwinding is inhibited. In support of this hypothesis, a cold-sensitive mutation in PRP44͞BRR2 (brr2-1) exacerbates (enhances) the U4-cs1 mutation (11).A selection for suppressors of the cold-sensitive growth defect of U4-cs1 yielded a mutation in the highly conserved 280-kDa splicing factor Prp8 (10). Prp8 is a U5 RNA-associated protein that con...

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mentioning

confidence: 84%

Distinct domains of splicing factor Prp8 mediate different aspects of spliceosome activation

Kuhn

Reichl

Brow

2002

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

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“…Subsequently, a large number of additional yeast genetic loci that suppress 3ЈSS splicing defects have been identified (Collins and Guthrie 1999;Siatecka et al 1999;Ben-Yehuda et al 2000b;Dagher and Fu 2001;Query and Konarska 2004). Scrutiny of sequence alignments of the affected residues has prompted us to split this domain in two.…”

Section: Nuclear Localization Signals (Nls)mentioning

confidence: 99%

Prp8 protein: At the heart of the spliceosome

Grainger¹,

Beggs²

2005

RNA

322

386

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Pre-messenger RNA (pre-mRNA) splicing is a central step in gene expression. Lying between transcription and protein synthesis, pre-mRNA splicing removes sequences (introns) that would otherwise disrupt the coding potential of intron-containing transcripts. This process takes place in the nucleus, catalyzed by a large RNA-protein complex called the spliceosome. Prp8p, one of the largest and most highly conserved of nuclear proteins, occupies a central position in the catalytic core of the spliceosome, and has been implicated in several crucial molecular rearrangements that occur there. Recently, Prp8p has also come under the spotlight for its role in the inherited human disease, Retinitis Pigmentosa.Prp8 is unique, having no obvious homology to other proteins; however, using bioinformatical analysis we reveal the presence of a conserved RNA recognition motif (RRM), an MPN/JAB domain and a putative nuclear localization signal (NLS). Here, we review biochemical and genetical data, mostly related to the human and yeast proteins, that describe Prp8's central role within the spliceosome and its molecular interactions during spliceosome formation, as splicing proceeds, and in post-splicing complexes. NOTE ON NOMENCLATUREIn this review, Prp8 and Prp8p represent the protein product of the wild-type PRP8 gene of Saccharomyces cerevisiae, while prp8-1 is an example of a mutant allele of the PRP8 gene. Human Prp8 protein is designated hPrp8, also known in the literature as PRPF8, PRPC8, p220, and 220K. In some places, to avoid confusion, yPrp8 is used to distinguish the yeast (S. cerevisiae) protein from the human form. sPrp8 SPP42 (Schmidt et al. 1999) or sPrp8 cwf6 (McDonald et al. 1999;Ohi et al. 2002) defines the ortholog in Schizosaccharomyces pombe. Confusingly, the cdc28 gene in S. pombe is also referred to as prp8 as a consequence of its role in both pre-mRNA splicing and cell cycle progression, and there is also a temperature-sensitive allele called prp8-1 that causes accumulation of pre-mRNA upon a shift to nonpermissive temperatures (Lundgren et al. 1996;Imamura et al. 1998). Cdc28 prp8 encodes a 112-kDa DEAH-box protein. This protein is not the ortholog of the U5 snRNP protein of S. cerevisiae that is discussed here.In discussions of pre-mRNA-protein cross-links or mutations that affect intron-exon junctions, 5ЈSS+2 refers to the second intronic base from the 5Ј end of the intron, 5ЈSS-2 is the penultimate base of the 5Ј exon, and 3ЈSS-2 is the penultimate base of the intron. PRE-mRNA SPLICINGPre-mRNA splicing involves two trans-esterification reactions within the highly dynamic spliceosome complex. A vast amount of mainly biochemical data led to a consensus view of an ordered pathway of spliceosome assembly that will be described in outline here (for further details, see Kramer 1996;Burge et al. 1999;Brow 2002). The small nuclear RNA-protein (snRNP) complexes, known as U1, U2, U4, U5, and U6 snRNPs, play key roles. U1 is the first snRNP to associate with pre-mRNA, interacting with the 5Ј splice site (5Ј...

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“…Prp8p is a large and highly conserved U5 snRNP component (Grainger and Beggs 2005) that interacts with numerous splicing factors (Whittaker et al 1990;Achsel et al 1998;Kuhn et al 1999Kuhn et al , 2002Stevens and Abelson 1999;BenYehuda et al 2000;Kuhn and Brow 2000;Gottschalk et al 2001;Stevens et al 2001;Wiesner et al 2002) and influences spliceosome assembly (Brown and Beggs 1992) as well as premRNA splice site selection (Umen andGuthrie 1995a,b, 1996;Konarska 1998;Dagher and Fu 2001). Furthermore, Prp8p interacts genetically and biochemically with all three splice sites in the pre-mRNA (Teigelkamp et al 1995a,b;Reyes et al 1996Reyes et al , 1999Collins and Guthrie 1999;Siatecka et al 1999;Query and Konarska 2004), which suggests a critical role during catalysis.…”

Section: Introductionmentioning

confidence: 99%

Ubiquitin binding by a variant Jab1/MPN domain in the essential pre-mRNA splicing factor Prp8p

Bellare¹,

Kutach²,

Rines³

et al. 2006

RNA

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The U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) are components of the spliceosome, which catalyzes pre-mRNA splicing. One of the largest and the most highly conserved proteins in the spliceosome is Prp8p, a component of the U5 snRNP. Despite its size and conservation, very few motifs have been identified that suggest specific biochemical functions. A variant of the Jab1/MPN domain found in a class of deubiquitinating enzymes is present near the C terminus of Prp8p. Ubiquitination regulates a broad range of cellular pathways, and its functions generally require ubiquitin recognition by one or more ubiquitin-binding domains (UBDs). No precise role for ubiquitin has been defined in the pre-mRNA splicing pathway, and no known UBDs have been found within splicing proteins. Here we show that a Prp8p fragment containing the Jab1/MPN domain binds directly to ubiquitin with an affinity comparable to other known UBDs. Several mutations within this domain that compromise splicing also reduce interaction of the fragment with ubiquitin-Sepharose. Our results define a new UBD and suggest functional links between ubiquitin and the pre-mRNA splicing machinery.

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

Cited by 29 publications

References 72 publications

Distinct domains of splicing factor Prp8 mediate different aspects of spliceosome activation

Distinct domains of splicing factor Prp8 mediate different aspects of spliceosome activation

Prp8 protein: At the heart of the spliceosome

Ubiquitin binding by a variant Jab1/MPN domain in the essential pre-mRNA splicing factor Prp8p

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