Mayerle Megan, Guthrie Christine
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143, USA.
RNA. 2016 May;22(5):793-809. doi: 10.1261/rna.055459.115. Epub 2016 Mar 11.
Pre-mRNA splicing must occur with high fidelity and efficiency for proper gene expression. The spliceosome uses DExD/H box helicases to promote on-pathway interactions while simultaneously minimizing errors. Prp8 and Snu114, an EF2-like GTPase, regulate the activity of the Brr2 helicase, promoting RNA unwinding by Brr2 at appropriate points in the splicing cycle and repressing it at others. Mutations linked to retinitis pigmentosa (RP), a disease that causes blindness in humans, map to the Brr2 regulatory region of Prp8. Previous in vitro studies of homologous mutations in Saccharomyces cerevisiaes how that Prp8-RP mutants cause defects in spliceosome activation. Here we show that a subset of RP mutations in Prp8 also causes defects in the transition between the first and second catalytic steps of splicing. Though Prp8-RP mutants do not cause defects in splicing fidelity, they result in an overall decrease in splicing efficiency. Furthermore, genetic analyses link Snu114 GTP/GDP occupancy to Prp8-dependent regulation of Brr2. Our results implicate the transition between the first and second catalytic steps as a critical place in the splicing cycle where Prp8-RP mutants influence splicing efficiency. The location of the Prp8-RP mutants, at the "hinge" that links the Prp8 Jab1-MPN regulatory "tail" to the globular portion of the domain, suggests that these Prp8-RP mutants inhibit regulated movement of the Prp8 Jab1/MPN domain into the Brr2 RNA binding channel to transiently inhibit Brr2. Therefore, in Prp8-linked RP, disease likely results not only from defects in spliceosome assembly and activation, but also because of defects in splicing catalysis.
前体mRNA剪接必须以高保真度和效率进行，才能实现正确的基因表达。剪接体利用DExD/H盒解旋酶来促进正常途径的相互作用，同时尽量减少错误。Prp8和Snu114（一种EF2样GTP酶）调节Brr2解旋酶的活性，在剪接循环的适当时间促进Brr2介导的RNA解旋，并在其他时间抑制其活性。与视网膜色素变性（RP，一种导致人类失明的疾病）相关的突变定位在Prp8的Brr2调节区域。先前对酿酒酵母中同源突变的体外研究表明，Prp8-RP突变体在剪接体激活方面存在缺陷。在这里，我们表明Prp8中的一部分RP突变也会导致剪接的第一步和第二步催化步骤之间的转换出现缺陷。虽然Prp8-RP突变体不会导致剪接保真度缺陷，但它们会导致剪接效率整体下降。此外，遗传分析将Snu114的GTP/GDP占据状态与Prp8对Brr2的依赖性调节联系起来。我们的结果表明，第一步和第二步催化步骤之间的转换是剪接循环中的一个关键位置，Prp8-RP突变体在这个位置影响剪接效率。Prp8-RP突变体位于连接Prp8 Jab1-MPN调节“尾部”与结构域球状部分的“铰链”处，这表明这些Prp8-RP突变体抑制Prp8 Jab1/MPN结构域向Brr2 RNA结合通道的调节性移动，从而暂时抑制Brr2。因此，在与Prp8相关的RP中，疾病可能不仅源于剪接体组装和激活的缺陷，还源于剪接催化的缺陷。
