Revealing molecular determinants of transcript-specific regulation in pre-mRNA splicing via rapid in vivo kinetic rate measurements

摘要 前信使RNA（pre-mRNA）剪接是基因表达的重要组成部分， 在真核生物中的表达，但在过去的十年里，我们对它的认识急剧增加， 调节基因表达的作用1.大多数高等真核生物，包括人类，调节选择性剪接， 作为蛋白质组扩展的工具，越来越多的人类疾病与突变有关 在这条路2，3.剪接体催化前体mRNA剪接的机制， 监管是一个复杂的问题，其解决方案仍然知之甚少，但对于理解监管至关重要。 许多疾病的病因。适当的调节需要剪接体忠实地组装在 在大量异常的“近同源”剪接位点的背景下激活“同源”剪接位点序列， 剪接体必须平衡这种高保真剪接位点选择与快速、有效剪接的需要。在 在最简单的层面上，对剪接体如何实现这种平衡的进一步了解将需要理解 两者：（1）剪接位点的顺式调控元件的景观，使它们能够被区分为 “同源”或“非同源”;和（2）剪接体区分这些位点的机制。 在这里描述的工作中，我们试图更好地理解前体mRNA剪接调控的基本机制 通过利用我实验室最近开发的一种强大的方法， 测序或MPE-seq。我们的方法是独特的，因为它允许全基因组检测前mRNA 剪接中间体。通过将该技术与由美国科学家开发的快速代谢RNA标记技术相结合， 其他人，我的小组现在已经确定了体内前mRNA剪接的两个化学步骤的速率， 芽殖酵母中剪接转录本的互补物。值得注意的是，这些数据显示， 率，无论是两个步骤之间的个人成绩单和不同的成绩单。的目标 这里描述的工作是利用从这些实验中获得的信息来推动我们的理解 这一规定的基本原则。

ABSTRACT It has long been known that pre-messenger RNA (pre-mRNA) splicing is an essential component of gene expression in eukaryotic organisms, yet the past decade has seen a dramatic increase in our appreciation for its role in regulating gene expression1. Most higher eukaryotes, including humans, regulate alternative splicing as a tool for proteome expansion, and an ever-increasing number of human diseases are associated with mutations in this pathway2,3. The mechanisms by which the spliceosome, which catalyzes pre-mRNA splicing, enacts this regulation is a complex problem whose solution remains poorly understood yet will be critical to understanding the etiology of many diseases. Proper regulation requires the spliceosome to faithfully assemble upon and activate ‘cognate’ splice site sequences in the background of scores of aberrant, ‘near-cognate’ splice sites, yet the spliceosome must balance this high fidelity splice site selection with the need for rapid, efficient splicing. At the simplest level, improved knowledge of how the spliceosome achieves this balance will require understanding both: (1) the landscape of cis-regulatory elements at splice sites that enable them to be distinguished as either ‘cognate’ or ‘non-cognate’; and (2) the mechanisms by which the spliceosome discriminates between such sites. In the work described here, we seek to better understand basic mechanisms of pre-mRNA splicing regulation by leveraging a powerful methodology recently developed in my lab called Multiplexed Primer Extension sequencing, or MPE-seq. Our approach is unique in that it allows for the genome-wide detection of pre-mRNA splicing intermediates. By combining this technique with rapid metabolic RNA labeling techniques developed by others, my group has now determined the in vivo rates of both chemical steps of pre-mRNA splicing across the complement of spliced transcripts in budding yeast. Remarkably, these data reveal a wide variation among the rates, both between the two steps for individual transcripts and between different transcripts. The goals of the work described here are to leverage the information derived from these experiments to push our understanding of the principles that underlie this regulation.