Functional association of essential splicing factor(s) with PRP19 in a protein complex.

Tarn, Woan‐Yuh; Hsu, Chi-Huei; Huang, Kuang-Tse; Chen, Hau-Ren; Kao, Hung‐Ying; Lee, Kuan‐Rong; Cheng, Soo‐Chen

doi:10.1002/j.1460-2075.1994.tb06527.x

Cited by 109 publications

(127 citation statements)

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“…Although many poly-ubiquitinated proteins are rapidly degraded by the 26S proteosome, ubiquitination can create a regulatory, rather than a proteolytic, signal 1 . Prp19p is essential for pre-mRNA splicing and is present in a complex that contains many proteins 9,26 . Thus far, however, its biochemical role in pre-mRNA splicing and that of the proteins with which it interacts are unknown.…”

Section: The U-box and Ubiquitinationmentioning

confidence: 99%

Structural insights into the U-box, a domain associated with multi-ubiquitination

Ohi

Kooi

Rosenberg

et al. 2003

Nat Struct Mol Biol

263

232

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The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the crossbrace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the Ubox. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val→Ile point mutation in the Prp19p U-box domain leads to premRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.Ubiquitin (Ub) targeting of proteins for degradation by the proteosome involves polyubiquination of substrate proteins via an enzyme cascade consisting of activating (E1), conjugating (E2) and ligating (E3) enzymes. E3 ubiquitin ligases vary widely in size, composition and enzymology, reflecting their regulatory role in substrate recognition 1 . HECT and RING finger domain-containing proteins constitute two classes of E3 ligases. HECT domains bind Ub through a thioester bond and transfer Ub directly to substrate. RING finger E3s facilitate the transfer of Ub from the E2 to the substrate, rather than binding Ub directly.Correspondence should be addressed to K.L.G. kathy.gould@mcmail.vanderbilt.edu or W.J.C. walter.chazin@vanderbilt.edu. 3 These authors contributed equally to this work. Competing interests statementThe authors declare that they have no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptA third class of E3 ubiquitin ligases has been recently identified 2,3 . This class of proteins contains a U-box motif, first identified in Saccharomyces cerevisiae Ufd2p. Ufd2p promotes the elongation of poly-ubiquitin chains in a U-box-dependent manner (recently termed an 'E4' activity) 4 . An alignment of U-boxes and RING motifs indicated that U-boxes lack the strictly conserved histidine and cysteine Zn 2+ -chelating residues found in RING fingers, but they share a similar pattern of hydrophobic and polar amino acids (Fig. 1a), raising the possibility that they have similar folds 5 .S. cerevisiae Prp19p is an essential pre-mRNA splicing factor that contains an N-terminal Ubox 6,7 . Interestingly, a mutation within the Prp19p U-box that results in the substitution of an isoleucine for a conserved valine (prp19-1) leads to pre-mRNA splicing defects and the disruption of several key protein-protein interactions within the spliceosome 8,9 . The profound physiological consequences that result from th...

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Section: The U-box and Ubiquitinationmentioning

confidence: 99%

Structural insights into the U-box, a domain associated with multi-ubiquitination

Ohi

Kooi

Rosenberg

et al. 2003

Nat Struct Mol Biol

263

232

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“…To isolate such a complex, the entire ORF of SNT309 was removed from a strain in which the Prp19p protein was tagged with the HA-epitope at its carboxyl terminus (28). The Prp19p-associated complex was isolated from extracts prepared from this strain by affinity chromatography on an anti-HA antibody-coupled Sepharose column (29). Components in the complex were examined by Far Western blotting for proteins that directly interact with Prp19p, and by Western blotting for components whose antibodies were available (27,37).…”

Section: Resultsmentioning

confidence: 99%

“…The procedure for Far Western blot analysis was modified from that described by Tarn et al (29), and in vitro phosphorylated Prp19p was used as a probe. The Prp19p protein was fused with an oligo-peptide (RREIL-SRRPSYRKD) of the protein kinase A site followed by the HA-epitope at its carboxyl terminus.…”

Section: Methodsmentioning

confidence: 99%

“…Prp19p seems to associate with the spliceosome concomitantly with or just after dissociation of U4 from the spliceosome (14,28), suggesting a possible role for mediating conformational rearrangement at this step of the spliceosome assembly process. Attempts to purify the Prp19p protein for functional studies led to the identification of a protein complex consisting of at least seven proteins associated with Prp19p (29). Among them, at least three can directly interact with Prp19p as analyzed by Far Western blotting (29).…”

mentioning

confidence: 99%

“…Attempts to purify the Prp19p protein for functional studies led to the identification of a protein complex consisting of at least seven proteins associated with Prp19p (29). Among them, at least three can directly interact with Prp19p as analyzed by Far Western blotting (29).…”

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Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing

Chen¹,

Tsao

Chen

et al. 1999

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

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The SNT309 gene was identified via a mutation that causes lethality of cells in combination with a prp19 mutation. We showed previously that Snt309p is a component of the Prp19p-associated complex and that Snt309p, like Prp19p, is associated with the spliceosome immediately after or concomitantly with dissociation of U4 from the spliceosome. We show here that extracts prepared from the SNT309-deleted strain (⌬SNT309) were defective in splicing but could be complemented by addition of the purified Prp19p-associated complex. Isolation of the Prp19p-associated complex from ⌬SNT309 extracts indicated that the complex was destabilized in the absence of Snt309p and dissociated on affinity chromatography, suggesting a role of Snt309p in stabilization of the Prp19p-associated complex. Addition of the affinity-purified Prp19p-Snt309p binary complex to ⌬SNT309 extracts could reconstitute the Prp19p-associated complex. Genetic analysis further suggests that Snt309p plays a role in modulating interactions of Prp19p with other associated components to facilitate formation of the Prp19p-associated complex. A model for how Snt309p modulates such interactions is proposed.Splicing of pre-mRNA takes place on a multicomponent ribonucleoprotein particle called the spliceosome, which consists of five small nuclear RNAs (snRNAs) and a number of protein factors (see refs. 1-8 for reviews). Numerous protein factors involved in the splicing reaction have been identified in mammals and yeast. Some are intrinsic components of the spliceosome (9-11), whereas others associate with the spliceosome only transiently (12)(13)(14). Many of the protein factors are components of the sn ribonucleoprotein particles (see refs. 1 and 15-18 for reviews).Yeast genetics provides a powerful tool for identification of protein factors involved in the splicing reaction, as well as for studying interactions between these components. A large number of PRP (precursor RNA processing) genes that encode protein splicing factors have been identified by screening temperature-sensitive mutants defective in pre-mRNA splicing (19,20). Other genes were identified through genetic interactions with introns, PRP genes, or snRNA genes (21-27). Over 40 genes that encode protein factors of the splicing machinery have been identified, but the precise functions of these proteins are not well understood.The yeast PRP19 gene was among the genes identified in a screen for temperature-sensitive mutants defective in splicing (20). Biochemical characterizations indicated that the Prp19p protein is essential for the pre-mRNA splicing reaction in vitro.

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The Use ofSaccharomyces cerevisiaeto Study the Mechanism of pre‐mRNA Splicing

Rymond

2012

Alternative Pre‐mRNA Splicing

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Functional association of essential splicing factor(s) with PRP19 in a protein complex.

Cited by 109 publications

References 69 publications

Structural insights into the U-box, a domain associated with multi-ubiquitination

Structural insights into the U-box, a domain associated with multi-ubiquitination

Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing

The Use ofSaccharomyces cerevisiaeto Study the Mechanism of pre‐mRNA Splicing

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