Characterizing the regulation of co-transcriptional splicing rates in human cells

摘要 选择性剪接在人类细胞类型中产生广泛的蛋白质多样性方面起着至关重要的作用。然而，在这方面， 选择性剪接的失调通常见于从发育障碍到 癌尽管在确定顺式作用序列和反式因子等特征方面取得了明显进展， 预测选择性剪接的结果，仍然缺乏对选择性剪接的关键机制的理解。 剪接调控例如，有强有力的迹象表明，剪接的动力学速率对于 替代性剪接决策;然而，由于当前测量剪接率方法的局限性， 这一概念尚未得到直接衡量。本提案的总体目标是调查 剪接动力学在更高的分辨率比以前可能的，并确定如何剪接速率调节， 人体细胞我假设剪接的动力学速率是决定基因表达的重要调控步骤。 选择性剪接结果。为了充分检验这一假设，有必要首先揭露是什么调节 体内剪接动力学。通过确定拼接速率的变化和调节，我将能够暴露 剪接动力学如何影响人类细胞中的选择性剪接决定。首先，我将揭示新的见解 通过探测50个组成型和50个非组成型基因的剪接率， 选择性剪接反应使用一种新的方法，测量剪接率在高分辨率。二我 将揭示剪接机制的不同组成部分如何使用剪接抑制剂控制剪接动力学 和剪接因子中具有基因突变的细胞系。第三，我将研究剪接增强子和 沉默子序列影响选择性剪接反应的动力学速率， 剪接动力学和选择性剪接决定之间的关系。最终，这项提案的结果将揭示 人类细胞中共转录剪接速率的变化比以前可能的更准确， 为剪接动力学如何调节和影响体内选择性剪接奠定了基础。

Abstract Alternative splicing has a vital role in generating extensive protein diversity across human cell types. However, misregulation of alternative splicing is commonly found in diseases ranging from developmental disorders to cancer. Despite clear progress in identifying the features, such as cis-acting sequences and trans factors, that predict alternative splicing outcomes, there remains a lack of understanding into key mechanisms of alternative splicing regulation. For instance, there is strong indication that the kinetic rate of splicing is crucial for alternative splicing decisions; however, due to limitations in current approaches for measuring splicing rate, this concept has yet to be directly measured. The overall goal of this proposal is to investigate the variation in splicing kinetics at higher resolution than previously possible and determine how splicing rate is regulated in human cells. I hypothesize that the kinetic rate of splicing is an important regulatory step in determining alternative splicing outcomes. In order to fully test this hypothesis, it is necessary to first expose what regulates splicing kinetics in vivo. By determining the variation and regulation of splicing rates, I will be able to expose how splicing kinetics influence alternative splicing decisions in human cells. First, I will uncover new insights into the variation of co-transcriptional splicing kinetics by probing the splicing rate of 50 constitutive and 50 alternative splicing reactions using a novel approach that measures splicing rates at high resolution. Second, I will reveal how different components of the splicing machinery control splicing kinetics using splicing inhibitors and cell lines with genetic mutations in splicing factors. Third, I will investigate how splicing enhancer and silencer sequences influence the kinetic rate of alternative splicing reactions in order to resolve a connection between splicing kinetics and alternative splicing decisions. Ultimately, the results from this proposal will reveal the variation in co-transcriptional splicing rates in human cells with more accuracy than previously possible and set the foundation for how splicing kinetics are regulated and influence alternative splicing in vivo.