Co-transcriptional mechanisms of neuronal microexon splicing: causes and consequences for 3' end processing

神经元微外显子剪接的共转录机制：3端加工的原因和后果

• 批准号: 10677614
• 负责人: Jackson Gordon
• 金额: $ 4.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677614
• 关键词: 5' Splice Site; Address; Alternative Splicing; Biology; Brain; Cells; Code; Codependence; Coupled; DNA-Directed RNA Polymerase; Data; Development; Engineering; Erythroid Cells; Event; Excision; Exons; Failure; Gene Expression Regulation; Genes; Genetic Enhancer Element; Genetic Transcription; Introns; Length; Libraries; Link; Messenger RNA; Methods; Molecular; Nervous System; Neurons; Neurotransmitters; Nucleotides; Patients; Play; Poly A; Polyadenylation; Polymerase; Positioning Attribute; Protein Isoforms; Proteins; RNA; RNA Polymerase II; RNA Processing; RNA Splicing; Regulation; Reporter; Role; Series; Signal Transduction; Site; Study models; System; Tail; Time; Tissues; Transcript; Transcription Elongation; Transcription Initiation Site; Variant; Work; Yeasts; autism spectrum disorder; cell type; experimental study; in vivo; mRNA Precursor; nervous system disorder; neuron development; novel; receptor; single molecule; transmission process

PROPOSAL SUMMARY/ABSTRACT Alternative splicing of pre-mRNA plays a key role in the regulation of gene expression and contributes to protein diversity between tissue types. Recent work has demonstrated that tissue-specific “microexons” that are 3-27 nucleotides in length are highly conserved in metazoans, play a prominent role in neuronal development, and are frequently mis-spliced in patients with autism spectrum disorder. Work from the last decade has demonstrated that splicing frequently occurs during transcription elongation (co-transcriptionally), with introns rapidly excised following their synthesis. It is unclear how microexons can be defined rapidly enough to be spliced during transcription elongation when both the 3' and 5' splice sites emerge from the RNA Polymerase II (Pol II) exit channel nearly simultaneously. Additionally, the co-dependence of microexon splicing with downstream mRNA processing events is unknown. The work outlined in this proposal, broken down into three independent aims, will fill substantial gaps in our understanding of neuronal microexon splicing regulation. In Aim 1, I will adapt sequencing-based methods pioneered in the Neugebauer lab to investigate the relationship between microexon splicing and transcription elongation. I hypothesize that microexons are spliced under different transcriptional parameters than “conventional” exons which are on average ten times longer. In Aim 2, I will analyze single molecules of mRNA to determine processing steps that co-occur in mRNAs with microexons. I anticipate that microexons will serve to influence downstream processing events (e.g polyA cleavage site choice). Finally, in Aim 3, I will use a series of splicing reporters to investigate the influence of local mRNA sequence to microexon splicing. This proposal addresses many unresolved questions related to neuronal microexon regulation.

提案摘要/摘要 前体 mRNA 的选择性剪接在基因表达调控中发挥着关键作用，并有助于蛋白质的形成 最近的研究表明，组织特异性“微外显子”为 3-27。 核苷酸长度在后生动物中高度保守，在神经元发育中发挥着重要作用，并且 过去十年的研究发现，自闭症谱系障碍患者的基因经常出现错误拼接。 证明剪接在转录延伸（共转录）过程中频繁发生，与内含子 目前还不清楚如何快速定义微外显子以进行剪接。 在转录延伸过程中，3' 和 5' 剪接位点均从 RNA 聚合酶 II (Pol II) 中出现 此外，微外显子剪接与下游的相互依赖性几乎同时存在。 该提案中概述的 mRNA 处理事件未知，分为三个独立的部分。 目标，将填补我们对神经元微外显子剪接调节理解的重大空白。 采用 Neugebauer 实验室首创的基于测序的方法来研究之间的关系 微外显子剪接和转录延伸。我领导认为微外显子是在不同的条件下进行剪接的。 转录参数比“传统”外显子平均长十倍。在目标 2 中，我会。 分析 mRNA 的单分子以确定 mRNA 与微外显子 I 共同发生的加工步骤。 预计微外显子将影响下游加工事件（例如多聚腺苷酸切割位点 最后，在目标 3 中，我将使用一系列剪接生成器来研究局部 mRNA 的影响。 该提案解决了与神经元相关的许多未解决的问题。 微外显子调节。