Determination of genome-wide splicing kinetics and their underlying regulation

项目摘要 mRNA的生物发生需要识别相对较小的外显子并将其拼接在一起， 在一片内含子序列中。大多数内含子的去除发生在转录水平上， 新生RNA复合物的移动目标。增加了这种复杂性，内含子并不总是被删除， 同样的方式。相反，选择性剪接可以产生具有不同功能的蛋白质，这些功能是由选择性剪接产生的。 相同的基因在过去的几十年里，人们发现了很多关于 复杂的分子机制调节剪接。然而，我们仍然知道很少的动力学， 剪接在其体内底物，新生的延伸前体mRNA上。大多数基因的共转录性质 剪接是为什么转录组范围的剪接动力学仍然是该领域进展的障碍的基础。 从新生RNA中去除内含子的速率被底物的转录动力学所混淆 本身为了确定共转录剪接速率，转录的动力学参数（例如， 起始速率、延伸速率和转录物切割速率）。在这里，我们揭开了一个 一种名为SKaTER-seq的新技术（剪接动力学和转录延伸率，通过 测序）和复杂的计算流水线，它们一起可以同时确定 转录和剪接速率的关键参数。我们的总体目标是应用 为了进一步了解剪接动力学是如何在细胞内发生的， 监管.第一个目标是确定转录和剪接动力学转录组范围内， 确定它们与基因组、染色质和反式作用因子的关系。第二个目标将决定 三种染色质组分（H3 K36 me 3，连接体组蛋白H1，组蛋白变体macroH 2A 1）在 转录和剪接动力学的建立。这些数据将阐明关键的未回答的问题， 关于染色质、转录延伸和剪接体扰动在定向中的作用的领域 拼接结果。

Project Summary The biogenesis of mRNA requires relatively small exons to be identified and stitched together from within an expanse of intronic sequence. The majority of intron removal occurs co-transcriptionally on the moving target of the nascent RNA complex. Adding to this complexity, introns are not always removed in the same way. Rather, alternative splicing can give rise to proteins with distinct functions arising from the same gene. Over the last few decades, a great deal has been uncovered about the mechanisms by which complex molecular machineries regulate splicing. However, we still know very little about the kinetics of splicing on its in vivo substrate, nascent elongating pre-mRNA. The co-transcriptional nature of most splicing underlies why transcriptome-wide splicing kinetics have remained a barrier to progress in the field. The rate of intron removal from nascent RNA is obfuscated by the transcriptional dynamics of the substrate itself. In order, to determine co-transcriptional splicing rates, the kinetic parameters of transcription (e.g. initiation rate, elongation rates and transcript cleavage rates) must also be determined. Here, we unveil a novel technique called SKaTER-seq (Splicing Kinetics and Transcription Elongation Rates through sequencing) and a sophisticated computational pipeline which together can simultaneously determine the critical parameters of transcription and splicing rates transcriptome-wide. Our overall goal is to apply the SKaTER-seq method in order to gain further mechanistic understanding of how splicing kinetics are regulated. The first aim will determine transcriptional and splicing kinetics transcriptome-wide and determine their relation to genomic, chromatin and trans-acting factors. The second aim will determine the role of three chromatin components (H3K36me3, linker histone H1, histone variant macroH2A1) in the establishment of transcriptional and splicing kinetics. This data will illuminate key unanswered questions in the field about the role of chromatin, transcriptional elongation, and spliceosomal perturbation in directing splicing outcomes.