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

反式因子结合和选择性剪接中的前 mRNA 内含子结构

• 批准号: 10275711
• 负责人: Lela Lynn Lackey
• 金额: $ 36.62万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10275711
• 关键词: Adenosine; Alternative Splicing; Antisense Oligonucleotides; Back; Binding; Binding Proteins; Binding Sites; Code; Complex; Derivation procedure; Disease; Elements; Excision; Exons; Feedback; Genes; Goals; Introns; Lead; Machine Learning; Mediating; Messenger RNA; Modeling; Nucleic Acid Regulatory Sequences; Process; Protein Isoforms; RNA; RNA Precursors; RNA Splicing; Reaction; Regulation; Regulatory Element; Research; Role; Site; Spliceosome Assembly Pathway; Spliceosomes; Structural Models; Structure; System; Test Result; Testing; Therapeutic; Transcript; Vision; Work; human disease; improved; interest; mRNA Precursor; novel; protein complex; tool

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.

项目摘要 剪接是从前体信使RNA（mRNA）中去除内含子区域并将内含子区域与内含子区域结合的过程。 外显子转化为成熟的转录本。选择性剪接导致差异内含子去除，产生多个 由共同的前体mRNA产生的可变剪接同种型。估计剪接的失调 至少有15%的人类疾病是由它引起的，而且可能导致更多的疾病。剪接反应是 剪接体是一种RNA蛋白复合物，分阶段组装到前体mRNA上。的 剪接体的最终活性受反式作用剪接体因子和顺式作用剪接体因子的组合影响。 前体mRNA中的元素。然而，参与顺式元件功能的因素，如序列基序， 和RNA结构折叠，还没有完全理解，并且大多数这样的元件仍然未被识别。这是 尤其是对于分支点选择，这是内含子内剪接体组装的重要早期阶段 在催化腺苷周围。虽然分支点被松散地认为是高度退化的序列， 基序，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 通过关注RNA结构介导的剪接体结合进行分支点选择 SF3B复合物为此，我将开发SF3B依赖的RNA二级结构模型。 前体RNA以鉴定富集的结构基序。我的工作将需要第一次大规模的内部推导 二级结构，包括分支点区域，这将有助于更好地理解RNA结构 顺式调节元件周围的基因影响剪接。我的第二个目标是开发一个识别CIS的系统 剪接调控元件并快速检测其功能意义。为了确定监管要素， 我将确定mRNA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相关的顺式调控区的新特征。的 这些位点对选择性剪接的功能影响将通过使用反义寡核苷酸进行实验测试。 在一些实施方案中，所述寡核苷酸（阿索）可以阻断或抑制调节区。我会建立一个积极的反馈循环 我可以预测顺式剪接调控元件，并立即测试它们对与ASO剪接的影响， 将测试结果返回到模型中以改进预测。该系统将导致准确的预测cis 在任何感兴趣的基因内的调节剪接元件。准确鉴定顺式元件将提高我们的 理解选择性剪接的协同调节的能力。我研究的长期目标是揭开 通过阐明RNA结构在剪接中的作用和开发一个强大的系统来识别剪接密码， 功能性顺式调节剪接元件并测试它们的活性。