Senn Katherine A, Lipinski Karli A, Zeps Natalie J, Griffin Amory F, Wilkinson Max E, Hoskins Aaron A
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 USA.
Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706 USA.
bioRxiv. 2024 Oct 21:2024.05.04.592262. doi: 10.1101/2024.05.04.592262.
Pre-mRNA splicing is catalyzed in two steps: 5' splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (1 and 2 step factors). We recently identified Fyv6 (FAM192A in humans) as a 2 step factor in ; however, we did not determine how widespread Fyv6's impact is on the transcriptome. To answer this question, we have used RNA-seq to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3' SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-EM structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only 2 step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the 1 step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3' SS.
前体mRNA剪接分两步催化：5'剪接位点（SS）切割和外显子连接。许多蛋白质与剪接体短暂结合，以特异性影响这些步骤（第一步和第二步因子）。我们最近鉴定出Fyv6（人类中的FAM192A）为第二步因子；然而，我们尚未确定Fyv6对转录组的影响有多广泛。为了回答这个问题，我们使用RNA测序来分析剪接变化。这些结果表明，Fyv6的缺失导致全转录组范围内非共识、分支点（BP）近端3' SS的激活。为了确定这些观察结果的分子基础，我们确定了一个含有Fyv6的酵母产物复合剪接体在2.3 Å分辨率下的高分辨率冷冻电镜结构。该结构表明，Fyv6是唯一与Prp22 ATP酶接触的第二步因子，并且Fyv6的结合与第一步因子Yju2的结合相互排斥。然后，我们利用这个结构剖析Fyv6功能域，并解释抑制突变体遗传筛选的结果。综合的转录组学、结构和遗传学研究使我们能够提出一个模型，其中Yju2/Fyv6交换促进外显子连接，而Fyv6促进共识、BP远端3' SS的使用。
