Pre-mRNA intronic structures in trans factor binding and alternative splicing

项目摘要 剪接是从前体信使RNA(Mrna)中去除内含子区域并结合的过程。 将外显子转化为成熟的转录。选择性剪接导致差异内含子的去除，产生多个 另一种剪接的异构体产生于共同的前体mRNA。剪接的失调被估计 至少占人类疾病的15%，而且很可能导致更多的疾病。剪接反应是 剪接体是一种RNA蛋白质复合体，分阶段组装到前体mRNA上。这个 剪接体的最终活性受到反式作用剪接体因子和顺式作用的组合的影响。 前体信使核糖核酸中的元件。然而，参与顺式元件功能的因素，如序列基序 和RNA结构折叠，目前还不完全清楚，其中大部分元素仍未确定。这是 对于分支点选择尤其如此，这是内含子内剪接体组装的关键早期阶段 围绕着催化腺苷。尽管分支点被松散地识别为高度退化的序列 Motif，它影响下游剪接位点的选择。我的第一个目标是阐明RNA结构的影响 剪接体结合RNA结构介导的分支点选择 关联的SF3B复合体。为此，我将开发依赖于SF3B的RNA二级结构模型 前体RNA，以确定丰富的结构基序。我的工作将涉及内含子的第一个大规模派生 二级结构，包括分支点区域，这将有助于更好地理解RNA的结构 在顺式调控元件周围，影响剪接。我的第二个目标是开发一个识别配置项的系统。 拼接调控元件，并快速测试它们的功能意义。为了识别监管要素， 我将确定mRNAs上的顺式特征，包括蛋白质结合位点、保守性和RNA二级结构， 并使用机器学习来发现功能上相关的顺式调节区域的新签名。这个 这些位点对选择性剪接的功能影响将通过使用反义来进行实验测试 能够阻断或抑制调控区域的寡核苷酸(ASO)。我将建立一个正反馈循环，在那里 我可以预测顺式剪接调控元件，并立即使用ASOS测试它们对剪接的影响， 测试结果返回到模型中，以改进预测。这一系统将导致对cis的准确预测。 任何感兴趣基因内的调控剪接元件。准确识别顺式元件将提高我们的 能够理解选择性剪接的共同调控。我的研究的长期愿景是揭开神秘面纱 通过阐明剪接密码中的RNA结构在剪接中的作用，并开发了一个强大的识别系统 功能顺式调控剪接元件并检测其活性。

Project Summary Splicing is the process of removing intronic regions from a precursor messenger RNA (mRNA) and combining exons into a mature transcript. Alternative splicing results in differential intron removal, producing multiple alternatively spliced isoforms arising from a common precursor mRNA. The dysregulation of splicing is estimated to underlie at least 15% of human diseases, and is likely to contribute to many more. Splice reactions are performed by the spliceosome, an RNA protein complex that is assembled onto precursor mRNA in stages. The final activity of the spliceosome is influenced by a combination of trans-acting spliceosome factors and cis elements within the precursor mRNA. However, factors involved in cis element function, like sequence motifs and RNA structure folds, are not fully understood, and a majority of such elements remain unidentified. This is particularly true for branchpoint selection, an essential early stage of spliceosome assembly within the intron around the catalytic adenosine. Although the branchpoint is loosely recognized as a highly degenerate sequence motif, it influences downstream splice site selection. My first goal is to elucidate the impact of RNA structure on branchpoint selection by focusing on RNA structure-mediated binding by the spliceosome associated SF3B complex. To do so, I will develop RNA secondary structure models for SF3B-dependent precursor RNAs to identify enriched structural motifs. My work will entail the first large-scale derivation of intronic secondary structures, including branchpoint regions, which will aid in better understanding of how RNA structures around cis regulatory elements influence splicing. My second goal is to develop a system to identify cis splicing regulatory elements and rapidly test their functional significance. To identify regulatory elements, I will identify cis features on mRNAs, including protein binding sites, conservation and RNA secondary structure, and use machine learning to discover novel signatures of functionally relevant cis regulatory regions. The functional impact of such sites on alternative splicing will be experimentally tested through use of antisense oligonucleotide (ASO) that can block or inhibit the regulatory region. I will set up a positive feedback loop where I can predict cis splice regulatory elements and immediately test their impact on splicing with ASOs, incorporating the test results back into the model to improve predictions. This system will lead to accurate prediction of cis regulatory splicing elements within any gene of interest. Accurate identification of cis elements will improve our ability to understand co-regulation of alternative splicing. The long-term vision of my research is to demystify the splicing code by clarifying the role of RNA structure in splicing and developing a powerful system to identify functional cis regulatory splicing elements and test their activity.