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

建议书摘要/摘要 Pre-mRNA的选择性剪接在基因表达的调控中起着关键作用，并对蛋白质有贡献 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 核苷酸的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 在自闭症谱系障碍患者中经常被错误拼接。过去十年的研究成果 证明剪接经常发生在转录延伸过程中(共转录)，与内含子 在它们合成后迅速切除。目前尚不清楚如何能够足够快地定义微外显子以进行剪接 在转录延伸过程中，3‘和5’剪接点都出现在RNA聚合酶II(POL II)上 几乎同时退出通道。此外，微外显子剪接与下游的相互依赖 信使核糖核酸的加工事件是未知的。这项提案中概述的工作分为三个独立的部分 目的，将填补我们在理解神经元微外显子剪接调控方面的实质性空白。在《目标1》中，我将 采用Neugebauer实验室首创的基于测序的方法来研究 微外显子剪接与转录延伸。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，我会 分析单分子信使核糖核酸，以确定与微外显子共同出现在信使核糖核酸中的加工步骤。我 预期微外显子将用于影响下游加工事件(例如，Polya裂解位点 选择)。最后，在目标3中，我将使用一系列剪接记者来研究局部mRNAs的影响 序列到微外显子的拼接。这项提案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。

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.