To produce messenger RNA, the spliceosome excises introns from precursor (pre)-mRNA and splices the flanking exons. To establish fidelity, the spliceosome discriminates against aberrant introns, but current understanding of such fidelity mechanisms is limited. Here we show that an ATP-dependent activity represses formation of mRNA from aberrant intermediates having mutations in any of the intronic consensus sequences. This proofreading activity is disabled by mutations that impair the ATPase or RNA unwindase activity of Prp22p, a conserved spliceosomal DExD/H-box ATPase. Further, cold-sensitive prp22 mutants permit aberrant mRNA formation from a mutated 3' splice-site intermediate in vivo. We conclude that Prp22p generally represses splicing of aberrant intermediates, in addition to its known ATP-dependent role in promoting release of genuine mRNA. This dual function for Prp22p validates a general model in which fidelity can be enhanced by a DExD/H-box ATPase.
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 ...
While even single nucleotide errors in pre‐mRNA splicing can lead to catastrophic consequences, our understanding of fidelity mechanisms in splicing is poor. Genetics studies have established the importance of fidelity mechanisms in splicing and implicated 3 of the 8 spliceosomal DExD/H box ATPases in competing with splicing to enable kinetic proofreading. However, due to a paucity of in vitro assays, it has remained unclear how specificity is established by kinetic proofreading and how rejected substrates are discarded. We have established in vitro that each chemical step in splicing is proofread by a DEAH box ATPase – 5′ splice site cleavage is proofread by Prp16 and exon ligation is proofread by Prp22. In a kinetic proofreading mechanism, specificity can be achieved if the rate of the ATPase and/or the rate of the proofread step varies with the authenticity of the substrate. We have found evidence that both Prp16 and Prp22 enhance specificity by discriminating against slowly splicing substrates. Both Prp16 and Prp22 reject substrates reversibly, necessitating an independent discard activity that we attribute to the DEAH box ATPase Prp43, which normally functions to discard a genuine intron after excision from the precursor. These findings establish that both chemical steps in splicing are proofread by a common ATP‐dependent framework and support a general role for DExD/H box ATPases in fidelity. Support: ACS, NIH.
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