Identification of novel splicing regulatory complexes

项目总结/摘要 前体mRNA剪接是真核生物基因表达的关键步骤，由大量的RNA- 识别前体mRNA上的短序列基序的结合蛋白（RBP）。这个过程在很大程度上 加上转录，许多内含子被删除，而主要的转录本， 通过RNA聚合酶II复合体与染色质结合。我们最近证明，前- mRNA结合的RNA结合Fox（Rbfox）蛋白质整合在具有确定的氨基酸序列的大蛋白质复合物中。 一组额外的剪接调节器，称为LASR。LASR综合体识别复合站点 由其几个成员的个体序列偏好决定。我们还观察到 LASR的调节活性，独立于该复合物的游离亚基的调节活性。 我们现在有证据表明，其他复合物含有不同的RNA结合蛋白 包括hnRNP A1、hnRNP A2/B1、SRSF 1、SRSF 2、ELAVL 1和RBM 10。我们还发现RBM 10 及其旁系同源蛋白RBM 5参与与17 S 核内有U2 snRNP。我们的研究结果为RBP相互作用提供了新的见解， 前体mRNA上调控复合物的组合组装。 我们建议纯化这些新的蛋白质复合物，表征它们的组成，并探测它们的结构。 内部结构。我们还将确定这些复合物在人类转录组中的天然靶点 并将研究它们的RNA募集位点与单个RBP亚基的RNA募集位点有何不同。 最后，我们将确定这些复合物在mRNA剪接中的活性以及 它们的蛋白质成分。 成功完成拟议的研究将对双方产生深远的影响 破译“剪接密码”，并了解遗传学背后的分子机制， 受剪接调节子影响的疾病。

Project Summary/Abstract Pre-mRNA splicing is a critical step in eukaryotic gene expression, controlled by a large set of RNA- binding proteins (RBPs) that recognize short sequence motifs on pre-mRNA. This process is largely coupled with transcription and many introns are removed while the primary transcript is this being associated with chromatin via the RNA polymerase II complex. We recently demonstrated that the pre- mRNA-bound RNA-binding Fox (Rbfox) proteins are integrated in a large protein complex with a defined set of additional splicing regulators, termed LASR. The LASR complex recognizes composite sites determined by the individual sequence preferences of several of its members. We also observed regulatory activities of LASR, separate from those of the free subunits of this complex. We now have evidence of additional complexes containing different sets of RNA-binding proteins including hnRNP A1, hnRNP A2/B1, SRSF1, SRSF2, ELAVL1, and RBM10. We further find RBM10 and its paralogous protein RBM5 to participate in novel interactions with components of the 17S U2 snRNP in the nucleus. Our findings offer new insights into the RBP interactions that drive the combinatorial assembly of regulatory complexes on pre-mRNA. We propose to purify these new protein complexes, characterize their composition, and probe their internal structure. We will also define the natural targets of these complexes in the human transcriptome and will examine how their RNA-recruitment sites differ from those of the individual RBP subunits. Finally, we will determine the activities of these complexes in mRNA splicing and the contributions of their protein constituents. Successful completion of the proposed research will have far-reaching implications for both deciphering the 'splicing code', and understanding the molecular mechanisms behind genetic disorders affected by splicing regulators.