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.

项目摘要 剪接是从前体信使RNA（mRNA）中去除内含子区域并组合的过程 外显子成一个成熟的成绩单。替代剪接会导致降低内含子的去除，从而产生多个 或者是由常见前体mRNA引起的剪接的同工型。估计剪接的失调 至少是15％的人类疾病的基础，并可能会促成更多人。剪接反应是 由剪接体进行，这是一种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反式剪接体因子和顺式的组合的影响 前体mRNA中的元素。但是，涉及顺式元素函数的因素，例如序列基序 RNA结构折叠，尚不完全了解，大多数此类元素仍然不明。这是 对于分支点选择，尤其是剪接体组装的必不可少的早期阶段 周围催化腺苷。尽管分支被松散地识别为高度退化的序列 主题，它影响下游剪接站点选择。我的第一个目标是阐明RNA结构的影响 通过关注剪接体的RNA结构介导的结合来选择分支点。 相关的SF3B复合物。为此，我将开发依赖SF3B的RNA二级结构模型 前体RNA鉴定富集的结构基序。我的工作将需要第一个大规模衍生的内含子派生 二级结构，包括分支区域，这将有助于更好地理解RNA结构 周围的CIS调节元件会影响剪接。我的第二个目标是开发一个系统来识别CI 拼接调节元件并迅速测试其功能意义。确定监管要素， 我将确定mRNA上的CIS特征，包括蛋白质结合位点，保护和RNA二级结构， 并使用机器学习发现功能相关的CIS调节区域的新型签名。这 这些站点对替代剪接的功能影响将通过使用反义进行实验测试 可以阻止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CIS剪接调节元素，并立即测试其对剪接的影响，并结合 测试结果重新回到模型中以改善预测。该系统将导致CIS的准确预测 任何感兴趣的基因内的调节剪接元素。准确识别顺式元素将改善我们的 了解替代剪接的共同调节的能力。我的研究的长期愿景是脱神秘 通过阐明RNA结构在拼接和开发强大的系统中的作用来识别剪接代码 功能性CIS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.