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: 10618260
• 负责人: Lela Lynn Lackey
• 金额: $ 36.56万
• 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/10618260
• 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; Machine Learning; Mediating; Messenger RNA; Modeling; Nucleic Acid Regulatory Sequences; Process; Productivity; Protein Isoforms; RNA; RNA Precursors; RNA Splicing; Reaction; Regulatory Element; Research; Role; Site; Spliceosome Assembly Pathway; Spliceosomes; Structure; System; Test Result; Testing; Therapeutic; Transcript; 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.

项目概要 剪接是从前体信使 RNA (mRNA) 中去除内含子区域并结合的过程 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 产生的选择性剪接异构体。估计剪接失调 是至少 15% 的人类疾病的根源，并且可能导致更多疾病。剪接反应是 该过程由剪接体执行，剪接体是一种 RNA 蛋白复合物，分阶段组装到前体 mRNA 上。这 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 mRNA 中的元件。然而，涉及顺式元件功能的因素，如序列基序 和 RNA 结构折叠尚未完全了解，并且大多数此类元件仍未被识别。这是 对于分支点选择尤其如此，分支点选择是内含子内剪接体组装的重要早期阶段 催化腺苷周围。尽管分支点被松散地认为是高度简并的序列 基序，它影响下游剪接位点的选择。我的首要目标是阐明 RNA 结构的影响 通过关注剪接体介导的 RNA 结构结合来选择分支点 相关的 SF3B 复合物。为此，我将开发 SF3B 依赖性的 RNA 二级结构模型 前体 RNA 来识别丰富的结构基序。我的工作将涉及内含子的首次大规模推导 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 围绕顺式调控元件影响剪接。我的第二个目标是开发一个识别顺式系统 剪接调控元件并快速测试其功能意义。为了确定监管要素， 我将识别 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监管区域的新特征。这 这些位点对选择性剪接的功能影响将通过使用反义进行实验测试 可以阻断或抑制调节区域的寡核苷酸（ASO）。我将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 ASO 剪接的影响，包括 将测试结果返回到模型中以改进预测。该系统将导致顺式的准确预测 任何感兴趣基因内的调节剪接元件。顺式元素的准确识别将提高我们的 理解选择性剪接的共同调控的能力。我研究的长期愿景是揭开神秘面纱 通过阐明 RNA 结构在剪接中的作用并开发强大的系统来识别剪接代码 功能性顺式调节剪接元件并测试其活性。

项目成果

How does precursor RNA structure influence RNA processing and gene expression?

• DOI: 10.1042/bsr20220149
• 发表时间: 2023-03-31
• 期刊: BIOSCIENCE REPORTS
• 影响因子: 4
• 作者: Herbert, Austin;Hatfield, Abigail;Lackey, Lela
• 通讯作者: Lackey, Lela

Global 5'-UTR RNA structure regulates translation of a SERPINA1 mRNA.

• DOI: 10.1093/nar/gkac739
• 发表时间: 2022-09-23
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Grayeski, Philip J.;Weidmann, Chase A.;Kumar, Jayashree;Lackey, Lela;Mustoe, Anthony M.;Busan, Steven;Laederach, Alain;Weeks, Kevin M.
• 通讯作者: Weeks, Kevin M.