Identification of novel splicing regulatory complexes

项目摘要/摘要 前信使核糖核酸剪接是真核基因表达的关键步骤，受一大套RNA调控。 识别Pre-mRNA上的短序列基序的结合蛋白(RBPs)。这个过程在很大程度上是 与转录结合，许多内含子被移除，而主要转录本是 通过RNA聚合酶II复合体与染色质结合。我们最近证明了前- MRNA结合的RNA结合狐狸(RBFox)蛋白被整合在一个大型蛋白质复合体中，该复合体具有明确的 一组额外的剪接调节器，称为LASR。LASR复合体识别复合站点 由它的几个成员的个别序列偏好决定。我们还观察到 LASR的调节活动，与这个复合体的游离亚单位的调节活动分开。 我们现在有证据表明，更多的复合体含有不同的RNA结合蛋白 包括hnRNP A1、hnRNP A2/B1、SRSF1、SRSF2、ELAVL1和RBM10。我们进一步发现RBM10 和它的同源蛋白RBM5参与与17S组分的新相互作用 U2SnRNP位于细胞核内。我们的发现为推动RBP相互作用提供了新的见解 调控复合体在前信使核糖核酸上的组合组装。 我们建议提纯这些新的蛋白质复合体，表征它们的组成，并探索它们的 内部结构。我们还将在人类转录组中定义这些复合体的自然靶点。 并将研究他们的RNA招募站点与单个RBP亚基的站点有何不同。 最后，我们将确定这些复合体在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”，理解基因背后的分子机制 受剪接调节器影响的疾病。

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.