Exon ligation is proofread by the DExD/H-box ATPase Prp22p

Mayas, Rabiah M.; Maita, Hiroshi; Staley, Jonathan P.

doi:10.1038/nsmb1093

Cited by 172 publications

(238 citation statements)

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“…In each case, mutant alleles that decrease ATPase activity are thought to allow more time for a limiting event preceding a conformational change. prp5 alleles improve splicing of branch region mutants by allowing more time for duplex formation with U2 snRNA (Xu and Query 2007), prp28 alleles improve splicing of 5 ′ SS mutants ), prp16 alleles improve substrates suboptimal for first-step catalysis by allowing a longer dwell time in the first-step conformation (Konarska and Query 2005;Villa and Guthrie 2005), and prp22 alleles improve splicing of substrates suboptimal for second-step catalysis (e.g., 3 ′ SS mutants) by allowing a longer dwell time in the second-step conformation (Mayas et al 2006). These common features were proposed (Burgess and Guthrie 1993) to similarly affect a preceding event, in each case limiting for different sets of (suboptimal) intron features or other spliceosomal interactions.…”

Section: Hsh155p/sf3b1 Mutations Alter Bs-u2 Duplex Fidelitymentioning

confidence: 99%

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SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing

et al. 2016

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Mutations in the U2 snRNP component SF3B1 are prominent in myelodysplastic syndromes (MDSs) and other cancers and have been shown recently to alter branch site (BS) or 3 ′ splice site selection in splicing. However, the molecular mechanism of altered splicing is not known. We show here that hsh155 mutant alleles in Saccharomyces cerevisiae, counterparts of SF3B1 mutations frequently found in cancers, specifically change splicing of suboptimal BS pre-mRNA substrates. We found that Hsh155p interacts directly with Prp5p, the first ATPase that acts during spliceosome assembly, and localized the interacting regions to HEAT (Huntingtin, EF3, PP2A, and TOR1) motifs in SF3B1 associated with disease mutations. Furthermore, we show that mutations in these motifs from both human disease and yeast genetic screens alter the physical interaction with Prp5p, alter branch region specification, and phenocopy mutations in Prp5p. These and other data demonstrate that mutations in Hsh155p and Prp5p alter splicing because they change the direct physical interaction between Hsh155p and Prp5p. This altered physical interaction results in altered loading (i.e., "fidelity") of the BS-U2 duplex into the SF3B complex during prespliceosome formation. These results provide a mechanistic framework to explain the consequences of intron recognition and splicing of SF3B1 mutations found in disease.

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Section: Hsh155p/sf3b1 Mutations Alter Bs-u2 Duplex Fidelitymentioning

confidence: 99%

“…Eight spliceosomal DExD/H ATPases remodel RNA and RNA-protein complexes and are essential for the progression of spliceosomal assembly and disassembly. In addition, they enhance splicing fidelity through kinetic proofreading (Burgess et al 1990;Mayas et al 2006;Xu and Query 2007;Koodathingal and Staley 2013).…”

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confidence: 99%

SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing

et al. 2016

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“…Prp16p, which promotes rearrangements required for exon ligation (14), discriminates against mutated branch site sequences at a later stage than Prp5p (10). Finally, Prp22p, which promotes release of the mRNA after exon ligation (15,16), discriminates against mutated consensus sites before exon ligation (11). Fidelity is also promoted by the sequestration of suboptimal substrates through equilibration between distinct spliceosomal states (17)(18)(19).…”

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confidence: 99%

“…For example, a substrate having a point mutation in an intronic consensus sequence can engage the spliceosome but fails to splice. Such substrates are discriminated against, in part, by at least three of the DExD/H box ATPases required to splice an optimal substrate (10)(11)(12). Specifically, Prp5p, which promotes binding of U2 to a substrate (13), discriminates against mutated branch site sequences (12).…”

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confidence: 99%

Spliceosome discards intermediates via the DEAH box ATPase Prp43p

Mayas

Maita²,

Semlow

et al. 2010

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

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150

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To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5′ exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product.RNA helicase | RNA processing | small nuclear RNA | ribonucleoprotein particle | Saccharomyces cerevisiae I n nuclear pre-mRNA splicing, the excision of introns is catalyzed by the spliceosome, a ribonucleoprotein machine comprising five snRNAs and ∼80 conserved proteins (for reviews, see ref. 1 and references therein). This machine assembles de novo on each pre-mRNA substrate and must rearrange its components throughout the splicing cycle through the activity of at least eight DExD/H box ATPases (2). The protein and RNA components of the spliceosome recognize the conserved elements of the intron at the 5′ splice site, the branch site, and the 3′ splice site. The RNA and possibly protein components also play key roles in catalyzing splicing, which occurs by two transesterification reactions (1). In the first reaction, the branch site adenosine attacks the 5′ splice site, generating a free 5′ exon and a lariat intermediate. In the second reaction, the 5′ exon attacks the 3′ splice site, excising the intron and ligating the exons. To establish specificity in splicing, the spliceosome discriminates optimal from suboptimal substrates.The specific pathway that discriminates against a suboptimal substrate depends on the extent to which a substrate is suboptimal. A grossly suboptimal substrate will fail to bind the spliceosome. Although such pre-mRNAs can be degraded in the nucleus (3), they can also be exported and then degraded in the cytoplasm (4-8). In contrast, optimal substrates are specifically retained in the nucleus to favor splicing (9).A nearly optimal substrate engages the spliceosome, necessitating more sophisticated fidelity ...

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“…Indeed, whether the ATP-dependent action of Prp22 leads to the release of other spliceosomal proteins is not clear. Prp22 has an additional ATP-independent role in promoting efficient step 2 catalysis in whole yeast cell extracts in vitro (Schwer and Gross 1998) and in contributing, by an ATP-dependent mechanism, to the fidelity of exon ligation (Mayas et al 2006).…”

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confidence: 99%

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

et al. 2013

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The spliceosome is a single-turnover enzyme that needs to be dismantled after catalysis to both release the mRNA and recycle small nuclear ribonucleoproteins (snRNPs) for subsequent rounds of pre-mRNA splicing. The RNP remodeling events occurring during spliceosome disassembly are poorly understood, and the composition of the released snRNPs are only roughly known. Using purified components in vitro, we generated post-catalytic spliceosomes that can be dissociated into mRNA and the intron-lariat spliceosome (ILS) by addition of the RNA helicase Prp22 plus ATP and without requiring the step 2 proteins Slu7 and Prp18. Incubation of the isolated ILS with the RNA helicase Prp43 plus Ntr1/Ntr2 and ATP generates defined spliceosomal dissociation products: the intron-lariat, U6 snRNA, a 20-25S U2 snRNP containing SF3a/b, an 18S U5 snRNP, and the ''nineteen complex'' associated with both the released U2 snRNP and intron-lariat RNA. Our system reproduces the entire ordered disassembly phase of the spliceosome with purified components, which defines the minimum set of agents required for this process. It enabled us to characterize the proteins of the ILS by mass spectrometry and identify the ATPase action of Prp43 as necessary and sufficient for dissociation of the ILS without the involvement of Brr2 ATPase.[Keywords: intron-lariat spliceosome; disassembly; Prp22; Prp43; Brr2] Supplemental material is available for this article. Pre-mRNA splicing proceeds by way of two phosphoester transfer reactions and is catalyzed by the spliceosome, which consists of the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) and numerous nonsnRNP proteins (Will and Lü hrmann 2011). The snRNPs are involved in recognizing short conserved sequences of the pre-mRNA, including the 59 and 39 splice sites (SS) and the branch site (BS), and in positioning the reactive nucleotides for catalysis. The spliceosome is a dynamic molecular machine, undergoing several major structural rearrangements during its functional cycle. These events are mediated by at least eight conserved DExD/H-box NTPases (hereafter termed RNA helicases) that act at discrete stages of the splicing pathway (Staley and Guthrie 1998;Cordin et al. 2012).Spliceosome assembly is initiated by the binding of U1 and U2 snRNPs to the 59 and the BS, respectively. This is followed by the recruitment of the U4/U6.U5 tri-snRNP, forming the precatalytic B complex. Catalytic activation of the spliceosome (leading to complex B act ) involves the displacement of U1 and U4 snRNAs from the spliceosome and the formation of new base pair interactions between the U6 and U2 snRNAs and the 59 SS (Staley and Guthrie 1998). The resulting RNA structure plays a central role in catalyzing both steps of splicing (Nilsen 1998).These RNA-RNA rearrangements are remodeled by the combined actions of the RNA helicases Prp28 and Brr2, which disrupt the base-pairing between U1 snRNA and the 59 SS and between the U4 and U6 snRNAs, respectively (Laggerbauer et al. 1998;Raghunathan and Guthrie 1998;Staley...

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Exon ligation is proofread by the DExD/H-box ATPase Prp22p

Cited by 172 publications

References 58 publications

SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing

SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing

Spliceosome discards intermediates via the DEAH box ATPase Prp43p

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

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