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: 10536025
• 负责人: Jackson Gordon
• 金额: $ 4.68万
• 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/10536025
• 关键词: 5' Splice Site; Address; Alternative Splicing; Biology; Brain; Cells; Code; Coupled; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Dependence; 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 structure; Neurons; Neurotransmitter Receptor; Nucleotides; Patients; Play; Polyadenylation; Positioning Attribute; Protein Isoforms; Proteins; RNA; 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; base; cell type; experimental study; in vivo; mRNA Precursor; nervous system disorder; neuron development; novel; 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起着关键作用，并有助于蛋白质的合成。 组织类型之间的多样性。最近的研究表明，组织特异性的3-27个“微外显子”， 核苷酸长度在后生动物中高度保守，在神经元发育中起重要作用， 在自闭症谱系障碍患者中经常发生错误拼接。过去十年的工作 表明剪接经常发生在转录延伸（共转录），内含子 在合成后迅速切除。目前还不清楚微外显子如何能够足够快地被定义以进行拼接 在转录延伸期间，当3'和5'剪接位点都从RNA聚合酶II（Pol II）出现时， 几乎同时退出渠道。此外，微外显子剪接与下游基因的相互依赖性， mRNA加工事件是未知的。本提案中概述的工作分为三个独立的部分， 目的，将填补我们对神经元微外显子剪接调控的理解的实质性空白。在目标1中，我将 采用纽介堡实验室开创的基于测序的方法来研究 微外显子剪接和转录延伸。我假设微外显子是在不同的 转录参数比“常规”外显子平均长十倍。在目标2中，我将 分析mRNA的单分子，以确定与微外显子共同发生在mRNA中的加工步骤。我 预期微外显子将用于影响下游加工事件（例如polyA切割位点 选择）。最后，在目标3中，我将使用一系列剪接报告子来研究局部mRNA的影响。 序列到微外显子剪接。这项提议解决了许多与神经元相关的悬而未决的问题。 微外显子调控