Snt309p, a Component of the Prp19p-Associated Complex That Interacts with Prp19p and Associates with the Spliceosome Simultaneously with or Immediately after Dissociation of U4 in the Same Manner as Prp19p

Chen, Hau-Ren; Jan, Shr-Peng; Tsao, Twee; Sheu, Yi-Jun; Banroques, Josette; Cheng, Soo‐Chen

doi:10.1128/mcb.18.4.2196

Cited by 48 publications

(61 citation statements)

References 69 publications

(85 reference statements)

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“…The prp19-1 point mutation encoding a V14I substitution 20 was identified in a genetic screen for splicing mutants 21 . Val14 is a part of the well-packed hydrophobic core of the Ubox (Fig.…”

Section: Prp19p Requires the U-box For Functionmentioning

confidence: 99%

“…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 this mutation underscore the importance of the U-box motif for Prp19p function and premRNA splicing.…”

mentioning

confidence: 99%

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

Ohi

Kooi

Rosenberg

et al. 2003

Nat Struct Mol Biol

261

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: Prp19p Requires the U-box For Functionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Ohi

Kooi

Rosenberg

et al. 2003

Nat Struct Mol Biol

261

232

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“…When Y2 and Y4 from the spliceosome formed in NTCdepleted extracts were digested with RNase H, each cross-linkcontaining fragment appeared as a smeared band (lanes [25][26][27][28][29][30][31][32][33][34][35][36], indicating that there might be multiple cross-linking sites on pre-mRNA and/or U5. Since cross-linking on the pre-mRNA was mapped near the 5Ј-end of the pre-mRNA, it is possible that the removal of half of U5 after RNase H digestion resulted FIG.…”

Section: Dynamicmentioning

confidence: 99%

The Prp19-associated Complex Is Required for Specifying Interactions of U5 and U6 with Pre-mRNA during Spliceosome Activation

Chan¹,

Cheng

2005

Journal of Biological Chemistry

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110

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Activation of the spliceosome involves a major structural change in the spliceosome, including release of U1 and U4 small nuclear ribonucleoprotein particles and the addition of a large protein complex, the Prp19-associated complex. We previously showed that the Prp19-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is released. Changes within the spliceosome upon binding of the Prp19-associated complex include remodeling of the U6/5 splice site interaction and destabilization of Lsm proteins to allow further interaction of U6 with the intron sequence. Here, we further analyzed interactions of U5 and U6 with pre-mRNA at various stages of spliceosome assembly from initial binding of tri-small nuclear ribonucleoprotein complex to the activated spliceosome to reveal stepwise changes of interactions. We demonstrate that both U5 and U6 interacted with pre-mRNA in dynamic manners spanning over a large region of U6 and the 5 exon sequences prior to the activation of the spliceosome. During spliceosome activation, interactions were locked down to small regions, and the Prp19-associated complex was required for defining the specificity of interaction of U5 and U6 with the 5 splice site to stabilize their association with the spliceosome after U4 is dissociated.Splicing of precursor mRNA takes place on a large, dynamic ribonucleoprotein complex, the spliceosome, which is constituted of five small nuclear RNAs, U1, U2, U4, U5, and U6, and numerous protein components (reviewed in Refs. 1-3). These components associate with pre-mRNA in a sequential manner to assemble the spliceosome into a functional complex, which can catalyze the splicing reaction (4). During spliceosome assembly, U1 first interacts with the 5Ј splice site, followed by association of U2 with the branch site to form a stable complex called the prespliceosome. The preformed U4/U6.U5 trisnRNP 1 is then recruited to the spliceosome to form a complex containing all five small nuclear RNAs. A subsequent structural rearrangement of the spliceosome releases U1 and U4, leading to the activation of the spliceosome.Base pair interactions play important roles in mediating splice site recognition and alignment by snRNPs in the spliceosome (reviewed in Refs. 1, 2, and 4). During spliceosome assembly, the 5Ј splice site is recognized by U1 in part via base pairing with the 5Ј-end of U1 RNA (5-7). Binding of U2 to the branch site is mediated by base pairing between U2 RNA and the branch site sequence (8, 9). The association of tri-snRNP with the spliceosome appears to also involve RNA-RNA interactions as demonstrated by isolation of cross-linked products of U4 small nuclear RNA to the second residue of the 5Ј splice site using a Saccharomyces cerevisiae in vitro trans-splicing system (10).Interactions of U5 and U6 with pre-mRNA in the spliceosome have been extensively studied. U5 interacts with exon sequences at the 5Ј and 3Ј splice sites through the conserved loop 1 sequence (11-16). The contacts between U5 and the 5Ј exon ...

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“…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)(22)(23)(24)(25)(26)(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.…”

mentioning

confidence: 99%

“…The SNT309 gene, which encodes a component of the Prp19p-associated complex, was identified in a genetic screen for mutations that exacerbate the phenotype of prp19 mutations (27). Cells disrupted in the SNT309 gene, although viable, are temperature-sensitive and accumulated pre-mRNA at the nonpermissive temperature.…”

mentioning

confidence: 99%

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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Snt309p, a Component of the Prp19p-Associated Complex That Interacts with Prp19p and Associates with the Spliceosome Simultaneously with or Immediately after Dissociation of U4 in the Same Manner as Prp19p

Cited by 48 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

The Prp19-associated Complex Is Required for Specifying Interactions of U5 and U6 with Pre-mRNA during Spliceosome Activation

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

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