Global control of co-transcriptional splicing

人类基因的选择性剪接(AS)非常普遍，并极大地扩展了蛋白质和RNA的谱系 来自人类基因组的产物。AS对于细胞分化和特性至关重要，它的 调节失调与一系列广泛且不断扩大的人类疾病有因果关系，包括肌肉疾病。 营养不良、神经退行性疾病和癌症。然而，我们目前对 在局部(基因)和全球(基因组范围)两个层面上对AS进行监管，因为缺乏提供 直接、高分辨率和定量地查看拼接过程。这一赤字反过来又阻碍了道路 了解剪接是如何调控赋予细胞特性和控制分化的进展 流程。平均每个人类基因的8个内含子是通过剪接体进行共转录处理的 与新生RNA中的特定序列结合的亚基和调节因子。这些顺式元素通常是 在内含子内，因此只在它们从RNA聚合酶出现时起作用，直到它们被剪接出来。 因此，为了剖析剪接调控机制，我们需要确定剪接发生的速度 以及新生转录产物中内含子的切割顺序。我们最近开发了CO-的纳米孔分析- 转录加工(Nano-CoP)，衡量剪接的动力学、顺序和协调 体内的内源基因。新生RNA被提纯，然后使用牛津纳米孔直接测序 获取长阅读的平台。我们发现剪接动力学受内含子长度和与 或者，剪接的外显子，该剪接顺序不遵循转录的顺序和相邻的 内含子有同时被配位剪接的倾向。这笔赠款的目标是确定 顺式作用元件和反式作用因子如何影响人类剪接动力学、剪接顺序和剪接 协调。具体目标1：确定反式作用因子如何影响剪接动力学。我们将学习八个 通过我们的分析或其他研究，与剪接调控有关的RNA结合蛋白。减少，减少 对于次要影响，我们将使用诱导降解系统在几个小时内降解目标因素。我们会 分别进行亚RNA-SEQ和纳米CoP检测，研究各因子丢失后的剪接动力学。具体目标2： 确定顺式作用元件在决定剪接动力学中的作用。我们将确定如何更改拼接 位点序列和其他顺式元件改变剪接动力学和选择性剪接。我们将使用CRISPR-CAS9 并利用自然遗传变异来研究顺式元件的扰动。具体目标3：确定 人类肌肉发生过程中剪接动力学与动脉硬化的关系。我们假设关键的交易行为 因子控制剪接动力学，进而影响AS。我们将研究剪接动力学如何在 使用纳米CoP的肌肉生成。肌肉发生剪接调节因子在控制剪接动力学中的作用 也被调查。总而言之，剪接动力学的变化将与结果相关，以确定 剪接如何通过剪接动力学进行调节的模型。

Alternative splicing (AS) of human genes is pervasive and greatly expands the repertoire of protein and RNA products arising from the human genome. AS is critical for cellular differentiation and identity, and its dysregulation has been causally linked with a broad and expanding array of human diseases, including muscular dystrophies, neurodegenerative disorders and cancers. However, we currently have limited insight into the regulation of AS at both the local (gene) and global (genome-wide) levels, due to a lack of tools that provide direct, high-resolution, and quantitative views into the splicing process. This deficit has in turn roadblocked progress in understanding how splicing is regulated to confer cellular identity and to control differentiation processes. The eight introns per average human gene are processed co-transcriptionally through spliceosomal subunits and regulatory factors binding to specific sequences in nascent RNA. These cis-elements are typically within introns and thus act only from when they emerge from RNA polymerase to when they are spliced out. Consequently, in order to dissect splicing regulation mechanisms, we need to determine how fast splicing occurs and the order of intron excision across nascent transcripts. We recently developed nanopore analysis of CO- transcriptional Processing (nano-COP) that measures the kinetics, order and coordination of splicing of endogenous genes in vivo. Nascent RNA is purified and then directly sequenced using the Oxford Nanopore platform to obtain long reads. We found that splicing kinetics is influenced by intron length and proximity to alternatively spliced exons, that splicing order does not follow the order of transcription and that neighboring introns have the propensity to be spliced coordinately at the same time. The goal of this grant is to determine how cis-acting elements and trans-acting factors impact human splicing kinetics, splicing order and splicing coordination. Specific Aim 1: Determine how trans-acting factors impact splicing dynamics. We will study eight RNA-binding proteins that are connected to splicing regulation by our analysis or other studies. To diminish secondary effects, we will use an inducible degradation system to degrade target factors within hours. We will perform subRNA-seq and nano-COP to study splicing dynamics after the loss of each factor. Specific Aim 2: Determine the role of cis-acting elements in dictating splicing dynamics. We will determine how changes to splice site sequences and other cis-elements alter splicing kinetics and alternative splicing. We will use CRISPR-Cas9 and leverage natural genetic variants to study perturbations to cis-elements. Specific Aim 3: Determine the relationship between splicing dynamics and AS during human myogenesis. We hypothesize that key trans-acting factors control splicing kinetics that in turn affect AS. We will study how splicing dynamics change during myogenesis using nano-COP. The roles of myogenesis splicing regulators in controlling splicing dynamics will also be investigated. In sum, changes in splicing kinetics will be associated with AS outcomes to determine models of how splicing is regulated by splicing dynamics.