Determination of genome-wide splicing kinetics and their underlying regulation

项目摘要 信使核糖核酸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 在一系列内含子序列中。大多数内含子的去除发生在共转录的 新生的RNA复合体的移动目标。增加这一复杂性的是，内含子并不总是在 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，这些蛋白质来自 同样的基因。在过去的几十年里，人们已经发现了许多关于 复杂的分子机制控制着剪接。然而，我们仍然对其动力学知之甚少。 在其体内的底物上剪接，新生的细长的Pre-mRNA。MOST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。 从新生rna中去除内含子的速度被底物的转录动力学混淆了。 它本身。为了确定共转录剪接率，转录的动力学参数(例如， 起始率、延伸率和转录本裂解率)也必须测定。在这里，我们揭开了一个 一种名为SICATER-SEQ(拼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 全基因组转录和剪接率的关键参数。我们的总体目标是将 滑冰-序贯方法，以进一步从机械上理解剪接动力学是如何进行的 受监管的。第一个目标将确定转录和剪接动力学在整个转录和 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 三种染色质成分(H3K36me3、连接物组蛋白H1、组蛋白变异体大分子H_2A1)在细胞周期调控中的作用 转录和剪接动力学的建立。这些数据将阐明以下关键悬而未决的问题 染色质、转录延长和剪接体干扰在导向中的作用 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.