Regulation and impact of alternative splicing in biology and disease

前-信使核糖核酸剪接是大多数后生动物基因表达所必需的基本过程。中的缺陷 剪接导致人类遗传病，剪接导致一些与生长控制有关的基因突变 与多种癌症有牵连。对前信使核糖核酸剪接基本机制的认识 因此，剪接位点识别是理解受调控的基因表达和人类 疾病。选择性剪接的控制是一个高度组合的过程，其中许多输入决定了 每个外显子的拼接结果。这些监管机制的一个关键特征是 反式作用剪接因子与顺式作用的RNA元件。我们使用高度集成的方法来调查 调控Pre-mRNA剪接的分子机制。这包括敲除和敲入纸巾 培养模型，使用放射性配基的重组分析，转录学，生物信息学，动力学，结构- 功能和生化技术。在未来五年，我们的目标是应对几个突出的挑战 在该领域，追求以下新颖的研究方向。(1)我们的演示表明，拼接监管 蛋白质表现出高度的位置依赖性活性，对剪接位点的选择产生负面或正面的影响 改变了我们对经典剪接激活因子(SR蛋白质)和经典剪接的看法 抑制子(HnRNP)。现在可以理解，U1 SnRNP的依赖于上下文的激活或抑制 作为网关，允许丰富的U1 SnRNP完成其剪接功能和保护 过早降解的前信使核糖核酸。然而，目前尚不清楚拼接监管机构是如何实现的 U1nRNP在5‘端剪接位的激活或抑制。我们的目标是剖析剪接的机制 通过采用多系统方法和了解U1 SNRNP构象在 介导剪接体组装。(2)内含子保留是一种重要的选择性剪接途径，它具有 没有进行广泛的研究。因此，它的监管并没有得到很好的理解。低效拼接的存在 编码外显子(Exitrons)中的内含子进一步强调了理解何时 内含子被有效地移除，当它们没有被移除时。我们将破译有效去除内含子的规则 并利用合成生物学方法研究顺式作用元件在这一过程中的影响。这个 争论是，在大规模平行报告分析中测试的序列变异的深度要大得多 而不是人类基因组提供的测试场景。在这里，我们将利用我们在 实验分子生物学和生物信息学。(3)人们普遍认识到，基因表达 事件是高度整合的，有证据表明，大多数前mRNA的加工发生在共同的 从转录上讲。这些步骤中的任何一个步骤的缺陷都与疾病有关。然而，大多数出版的 研究只评估基因表达的稳定水平，或者只关注一个步骤。这会忽略 基因表达步骤的动态，这些步骤共同有助于从mRNAs中产生蛋白质。 因此，目前尚不清楚rna加工动力学和mrna稳定性如何转化为终点基因。 表达式签名。我们已经建立了一种可靠的代谢标记新生RNA的方法，它 使我们能够追踪从合成到降解的转录本。这个项目的工作将探索如何稳定状态 MRNA水平已经确定，剪接和翻译调节因子SRSF1如何影响mRNA动态 以及当细胞经历转变时这些过程是如何调整的。我们研究计划的目标是 对外显子识别和选择性剪接有更好的理解。新的机械论洞察力将 被利用来改进策略，以治疗性地针对这一关键的基因表达步骤。

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in splicing lead to human genetic disease, and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer. Insights into the basic mechanisms of pre-mRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. The control of alternative splicing is a highly combinatorial process, where many inputs dictate the splicing outcome for each exon. A critical feature of these regulatory mechanisms is the specific interaction of trans-acting splicing factors with cis-acting RNA elements. We use a highly integrated approach to investigate the molecular mechanisms that regulate pre-mRNA splicing. This includes knockout and knock-in tissue culture models, reconstitution assays using radioligands, transcriptomics, bioinformatics, kinetics, structure- function and biochemical techniques. In the next five years, we aim to address several outstanding challenges in the field, pursuing the following novel research directions. (1) Our demonstration that splicing regulatory proteins display highly position-dependent activities that negatively or positively influence splice site choice changed the way we think about the classical splicing activators (SR proteins) and the classical splicing repressors (hnRNPs). It is now appreciated that the context-dependent activation or repression of U1 snRNP serves as a gateway to allow the abundant U1 snRNP to fulfill its splicing function and its role to protect the pre-mRNA from premature degradation. However, it is not understood how splicing regulators achieve activation or repression of U1snRNP at the 5’ splice site. We aim to dissect the mechanisms of splicing repression by embracing multi-system approaches and by understanding the role of U1 snRNP conformers in mediating spliceosomal assembly. (2) Intron retention is an important alternative splicing pathway that has eluded extensive study. Thus, its regulation is not well-understood. The existence of inefficiently spliced introns within coding exons (exitrons) further highlights the biological importance of understanding when introns are removed efficiently and when they are not. We will decipher the rules of efficient intron removal and investigate the impact of cis-acting elements in this process using synthetic biology approaches. The argument is that the depth of the sequence variation tested in massively parallel reporter assays is far greater than the testing landscape that the human genome offers. Here, we will take advantage of our expertise in experimental molecular biology and bioinformatics. (3) It has become widely appreciated that gene expression events are highly integrated, with evidence suggesting that most pre-mRNA processing occurs co- transcriptionally. Defects in any one of these steps has been linked to disease. However, most published studies evaluate only steady-state levels of gene expression or focus only on a single step. This ignores the dynamics of gene expression steps that collectively contribute to the generation of proteins from mRNAs. Thus, it is unclear how the kinetics of RNA processing and mRNA stability translate into an endpoint gene expression signature. We have established a reliable method to metabolically label nascent RNA, which allows us to track transcripts from synthesis to degradation. Work in this project will probe how steady state mRNA levels are established, how the splicing and translation regulator SRSF1 influences mRNA dynamics and how these processes adjust as a cell undergoes transformation. The goals of our research program are to obtain a better understanding of exon recognition and alternative splicing. The new mechanistic insights will be leveraged to improve strategies to therapeutically target this essential gene expression step.