Mayas Rabiah M, Maita Hiroshi, Semlow Daniel R, Staley Jonathan P
Graduate Program in Biochemistry and Molecular Biophysics, University of Chicago, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2010 Jun 1;107(22):10020-5. doi: 10.1073/pnas.0906022107. Epub 2010 May 12.
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.
为了提高核前体mRNA剪接的忠实性，剪接体能够识别并丢弃那些已与剪接体结合但并不理想的底物。虽然DExD/H盒ATP酶被认为与丢弃不理想的底物有关，但丢弃不理想底物的机制仍不清楚。有确凿证据表明，不理想的、发生突变的套索状中间体可以被转运到细胞质中进行周转，我们发现核糖体能够翻译发生突变的套索状中间体。通过甘油梯度分析，我们发现剪接体能够在体内以一种需要DEAH盒ATP酶Prp43p的方式解离发生突变的套索状中间体。通过体外实验，我们证明Prp43p能不加区分地促进不理想和理想的5'外显子及套索状中间体的丢弃。最后，我们证明在抑制隐蔽剪接位点的剪接过程中需要Prp43p。我们提出了一个外显子连接忠实性的模型，其中DEAH盒ATP酶Prp22p减缓不理想中间体通过外显子连接的流动，而Prp43p通常促进中间体的丢弃，从而建立了一个停滞中间体周转的途径。由于Prp43p在剪接外显子后也促进剪接体的解体，这项工作在不理想中间体的丢弃和真正切除的内含子产物的解离之间建立了一种平行关系。
