The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5′ splice site

Reyes, José Luis; Gustafson, E. Hilary; Luo, Hongbo; Moore, Melissa J.; Konarska, Maria M.

doi:10.1017/s1355838299981785

Cited by 90 publications

(98 citation statements)

References 34 publications

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“…The p220 that cross-linked to benzophenone at position −7 is most likely hPrp8p, as we and others have previously demonstrated that hPrp8p cross-links strongly near the end of the 5Ј exon and at the 5Ј splice site (Chiara et al 1996 and references therein; Reyes et al 1999;Le Hir et al 2000a). Likewise, the p170 that cross-linked only after exon ligation may well be SRm160-using a different antiserum from that used here (see Materials and Methods), we have previously shown that SRm160 can become cross-linked to benzophenone incorporated adjacent to the exon-exon junction of spliced mRNA (Le Hir et al 2000a).…”

Section: Enhanced 5ј Exon Association Of Ref/aly and Srp20 Upon Exon supporting

confidence: 51%

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

Reichert

Hir²,

Jurica³

et al. 2002

Genes Dev.

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126

137

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A general consequence of pre-mRNA splicing is the stable deposition of several proteins 20-24 nucleotides (nt) upstream of exon-exon junctions on spliced mRNAs. This exon junction complex (EJC) contains factors involved in mRNA export, cytoplasmic localization, and nonsense-mediated mRNA decay. Here we probed the mechanism and timing of EJC assembly. Over the course of splicing, the 5 exon is subject to numerous dynamic protein-RNA interactions involving at least nine distinct polypeptides. Within the fully assembled spliceosome, these interactions afford protection to the last 25-27 nt of the 5 exon intermediate. Throughout their lifetimes in vivo, mRNAs exist as mRNA-protein particles (mRNPs). The associated proteins control every aspect of mRNA metabolism, from subcellular transport to translational efficiency to rate of decay. Exactly which proteins associate with a particular mRNA depends on its sequence, its subcellular localization, and its synthetic history. Furthermore, the complement of mRNP proteins evolves as the mRNA moves to different locations and is acted on by such processes as nuclear export and translation. Many proteins join the mRNP in the nucleus and then accompany it to the cytoplasm, where they influence subsequent mRNA metabolism ( These splicing-specific mRNP proteins constitute the exon junction complex (EJC), which associates with spliced mRNAs in a sequence-independent manner a fixed distance upstream of exon-exon junctions (Le Hir et al. 2000b). Using coimmunoprecipitation strategies, we and others recently reported that the EJC assembled in HeLa nuclear extract contains numerous epitopes, including those recognized by antibodies against REF/Aly, Y14, SRm160, DEK, RNPS1, UAP56, and Magoh (Blencowe et al. 1998;Mayeda et al. 1999;Kataoka et al. 2000Kataoka et al. , 2001Le Hir et al. 2000a,b, 2001bMcGarvey et al. 2000;Zhou et al. 2000;Gatfield et al. 2001;Luo et al. 2001). When assembled in vivo, the EJC is additionally precipitated by antibodies against Upf2, Upf3, and the TAP:p15 heterodimer (Kim et al. 2001b;Le Hir et al. 2001a;Lykke-Andersen et al. 2001). Finally, REF/Aly, Y14, SRm160, RNPS1, Upf2, Upf3, and TAP were all present in mRNPs immunopurified from the nuclear fraction of COS cells with antibodies against a subunit of the nuclear cap binding complex (Lejeune et al. 2002).The known EJC components function at multiple levels of mRNA metabolism. SRm160 and RNPS1 were originally characterized as activators of pre-mRNA splicing (Blencowe et al. 1998;Mayeda et al. 1999

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Section: Enhanced 5ј Exon Association Of Ref/aly and Srp20 Upon Exon supporting

confidence: 51%

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

Reichert

Hir²,

Jurica³

et al. 2002

Genes Dev.

Self Cite

126

137

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“…2D). Furthermore, in HeLa extract, 5Ј ss has been observed to cross-link to a region close to residues 1,894-1,898 (corresponding to residues 1,966-1,970 in yPrp8) (32). Residues S1966, A1967, and S1970 (corresponding to Q1894, A1895, and K1898 in hPrp8) are indeed surface exposed in the ␤-finger domain structure, although two of the above residues are not conserved in yPrp8 (Fig.…”

Section: Discussionmentioning

confidence: 97%

Crystal structure of the β-finger domain of Prp8 reveals analogy to ribosomal proteins

Yang

Zhang

et al. 2008

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

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Prp8 stands out among hundreds of splicing factors as a key regulator of spliceosome activation and a potential cofactor of the splicing reaction. We present here the crystal structure of a 274-residue domain (residues 1,822-2,095) near the C terminus of Saccharomyces cerevisiae Prp8. The most striking feature of this domain is a ␤-hairpin finger protruding out of the protein (hence, this domain will be referred to as the ␤-finger domain), resembling many globular ribosomal proteins with protruding extensions. Mutations throughout the ␤-finger change the conformational equilibrium between the first and the second catalytic step. Mutations at the base of the ␤-finger affect U4/U6 unwinding-mediated spliceosome activation. Prp8 may insert its ␤-finger into the first-step complex (U2/U5/U6/pre-mRNA) or U4/U6.U5 tri-snRNP and stabilize these complexes. Mutations on the ␤-finger likely alter these interactions, leading to the observed mutant phenotypes. Our results suggest a possible mechanism of how Prp8 regulates spliceosome activation. These results also demonstrate an analogy between a spliceosomal protein and ribosomal proteins that insert extensions into folded rRNAs and stabilize the ribosome. P re-mRNA splicing is a critical step for gene expression in all eukaryotes. In eukaryotes, DNA is first transcribed to premRNAs whose introns have to be accurately removed before mRNA export and translation. Introns are removed through two transesterification steps. In the first step, the 2Ј-OH group of a critical adenosine residue in the branch point sequence (BPS) attacks the 5Ј end of the intron and forms a lariat intermediate. In the second step, the newly freed 3Ј-OH group of the 5Ј-end exon attacks the 3Ј-end of the intron, releasing the lariat and ligating the two exons.Pre-mRNA splicing is catalyzed by the spliceosome, a large RNA/protein complex that contains five snRNAs (U1, U2, U4, U5, and U6) and over 100 different protein factors. The spliceosome appears to assemble on pre-mRNA in a stepwise manner (1), although evidence also exists that the spliceosome may preassemble before encountering a pre-mRNA substrate (2). During spliceosome assembly, the 5Ј splice site (ss), BPS, and 3Ј ss of pre-mRNA are first recognized by the U1 snRNP, SF1/BBP, and U2AF65/35, respectively. Next, U2 snRNP replaces SF1 and base-pairs with the BPS. Subsequently, the U4/U6.U5 tri-snRNP joins the spliceosome. Next, extensive structural rearrangements occur to form the catalytically active spliceosome complex (first-step complex), which contains U2, U5, U6, and the pre-mRNA (3). During this activation process, the base-pairing between the 5Ј ss and U1 snRNA is disrupted, and the 5Ј ss interacts with the ACAGA box of U6 instead, using largely the same nucleotides that base-paired with U1 snRNA. The base-pairing between U4 and U6 is also disrupted, and new interactions between U2 and U6, which are mutually exclusive with those in the original U4/U6 complex, are formed. In addition, the BPS interacts with U2 snRNA, and both exons interac...

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“…The most likely candidate for an active-site protein in the spliceosome is Prp8. Prp8 has been cross-linked to the 5Ј splice site and appears to functionally interact with both splice sites during splicing (23,24). Recent crystallographic data show that a region of Prp8 that cross-links to the 5Ј splice site folds into an RNase H domain (25)(26)(27), which is remarkable, because RNase H domains catalyze the cleavage of phosphodiester bonds in RNA by using a 2-metal-ion mechanism (28).…”

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confidence: 99%

The spliceosome as ribozyme hypothesis takes a second step

Butcher

2009

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

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T he spliceosome is a massive assembly of 5 RNAs and many proteins that, together, catalyze precursor-mRNA (pre-mRNA) splicing. The splicing mechanism involves 2 steps: (i) cleavage of the 5Ј exon-intron phosphodiester bond and formation of a new 2Ј-5Ј bond resulting in an intron ''lariat,'' and (ii) exon ligation (Fig. 1A). This 2-step phosphoryl transfer mechanism is suspiciously identical to the reaction catalyzed by the group II self-splicing introns, which are ribozymes. The precedent for a ribozymecatalyzed splicing mechanism, combined with the fact that the spliceosome has an RNA core that has been highly conserved for Ͼ1 billion years, led researchers to hypothesize that the spliceosome may use an RNA-based catalytic mechanism (1). However, the spliceosome as ribozyme hypothesis has been exceedingly difficult to prove, for 2 major reasons. First, the spliceosome contains many proteins that are essential for splicing (2). Second, the uncatalyzed rate of RNA ligation in vitro is significant when measured over many hours (3). This latter point makes it difficult to establish whether an inefficient reaction is actually caused by a catalytic rate enhancement or occurs spontaneously because of template-driven proximity. This is a particular concern for 1-step phosphoryl transfer reactions. Furthermore, it is possible to serendipitously obtain unrelated activities from RNAs, perhaps because a significant region of RNA conformational space retains some degree of catalytic activity (4). Therefore, it is important to be very cautious when interpreting an inefficient RNA-based reaction. The relevance of using engineered, protein-free RNA systems to study splicing has been recently argued (5, 6). In a recent issue of PNAS, Valadkhan et al. (7) demonstrated that an RNA complex derived from the spliceosome can perform a reaction that resembles splicing (Fig. 1B). This finding is significant because the reaction appears to be identical to the second step of splicing and is Ϸ10-fold more efficient than previous studies that used similar ).An interesting aspect of this new reaction is that the first step seems to be generated via hydrolysis rather than by a 2Ј-5Ј branching reaction (Fig. 1B). This finding is different from spliceosomecatalyzed pre-mRNA splicing (Fig. 1 A), but has precedence among the group II ribozmes (11). The second step of the reaction, however, results in exon ligation and formation of a new 5Ј-3Ј bond, just as in splicing. This reaction also has sequence requirements that closely parallel those of the spliceosome and requires magnesium. These data support, but do not prove, the idea that the reaction is related to pre-mRNA splicing. In future experiments, it will be important to investigate this reaction further for its mechanistic parallels to splicing. Currently, it is not clear how the substrate RNAs are coordinated in the reaction, and whether or not the system faithfully recapitulates the interactions in the spliceosome remains to be determined. Another important question concerns how the cata...

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The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5′ splice site

Cited by 90 publications

References 34 publications

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

Crystal structure of the β-finger domain of Prp8 reveals analogy to ribosomal proteins

The spliceosome as ribozyme hypothesis takes a second step

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