Regulation of mRNA isoform fate

描述（由申请人提供）： 选择性剪接（AS）过程是从蛋白质编码基因产生mRNA多样性的强大机制。来自相同基因的替代mRNA同种型可以彼此微妙地不同，或者在它们的序列组成中具有根本的改变。值得注意的是，对于这种多样的mRNA库是否转化为蛋白质知之甚少。解决这个问题的一个有趣的线索来自我们以前的研究，表明选择性剪接的过程影响了所得mRNA异构体的翻译效率。在这个建议中，我们使用全基因组和分子方法来解开复杂的机制耦合转录后基因表达。为了实现这一目标，我们提出了以下具体目标：（1）确定AS-TC在人类基因调控中的流行和影响。在这个目标中，我们将量化mRNA异构体的翻译多聚核糖体在15个不同的ENCODE细胞系和两个不同的灵长类动物干细胞模型系统，使用我们最近开发的Frac-Seq方法。这些实验将揭示AS-TC调节事件的范围，并确定这是否是基因表达的空间或时间调节的保守机制。(2)以富含丝氨酸和精氨酸的蛋白SRSF 1为模型，我们将阐明穿梭RNA结合蛋白在转录后基因调控偶联中的作用和靶点。为此，我们将利用CLIP-Seq定位SRSF 1或非穿梭突变体在不同亚细胞组分中的结合位点;利用Frac-Seq确定AS-TC对SRSF 1的依赖性;利用RNA亲和层析发现新的反式作用AS-TC因子。这项工作的结果将为穿梭蛋白如SRSF 1在转录后基因表达协调中的功能定义一个新的范例。(3)确定调节亚型特异性mRNA翻译的分子机制。在这一目标中，我们将阐明使用FragSeq和QUEPASA调节多核糖体缔合的RNA元件和结构的完整库，然后以生物化学方式确定AS-TC中涉及的序列元件如何调节翻译起始和延伸的机制。通过确定AS-TC元件如何调节mRNA翻译，我们将能够反驳或支持这一提议的中心假设，即选择性剪接产生具有不同翻译效率的异构体。如果与差异多聚核糖体结合相关的序列不直接控制翻译起始或延伸，那么我们将 考虑我们的另一种假设：mRNA异构体多样性的显著部分是由 从嘈杂的剪接，然后排除从多聚核糖体的一些未知的mRNA监视途径。解决这一重要问题不仅将揭示顺式元件如何影响翻译产量，而且还将定义选择性剪接和mRNA翻译过程之间的机制联系。从长远来看，我们的研究计划将为基于RNA的诊断和治疗提供新的机会，这些诊断和治疗将适用于各种人类疾病。

DESCRIPTION (provided by applicant): The process of alternative splicing (AS) is a powerful mechanism for generating mRNA diversity from protein coding genes. Alternative mRNA isoforms from the same gene can differ subtly from each other or have radical alterations in their sequence composition. Remarkably, very little is known about whether or not this diverse pool of mRNA is converted to protein. One intriguing clue for solving this problem comes from our previous studies suggesting that the process of alternative splicing influences the translational efficiency of the resultant mRNA isoforms. In this proposal, we use both genome-wide and molecular approaches to untangle the intricate mechanisms coupling post-transcriptional gene expression. To accomplish this goal we propose the following specific aims: (1) Determine the prevalence and impact of AS-TC in human gene regulation. In this aim we will quantify mRNA isoforms with respect to the translating polyribosomes in 15 different ENCODE cell lines and two different primate stem cell model systems using our recently developed Frac-Seq methodology. These experiments will reveal the scope of AS-TC regulatory events and determine if this is a conserved mechanism for spatial or temporal regulation of gene expression. (2) Using the Serine and Arginine-rich protein SRSF1 as a model, we will elucidate the roles and targets of shuttling RNA binding proteins in coupling of post- transcriptional gene regulation. In this aim we will map the binding sites of SRSF1 or non-shuttling mutants in different subcellular fractions using CLIP-Seq; Determine the dependency of AS-TC on SRSF1 using Frac-Seq; Discover novel trans-acting AS-TC factors using RNA affinity chromatography. The results of this work will define a new paradigm for the function(s) of shuttling proteins such as SRSF1 in the coordination of post- transcriptional gene expression. (3) Determine the molecular mechanisms regulating isoform-specific mRNA translation. In this aim we will elucidate the full repertoire of RNA elements and structures that regulate polyribosome association using FragSeq and QUEPASA then determine biochemically how sequence elements involved in AS-TC modulate mechanisms of translation initiation and elongation. By determining how AS-TC elements regulate mRNA translation we will be able to refute or support the central hypothesis of this proposal, that alternative splicing generates isoforms with different translational efficiency. If sequences associated with differential polyribosome association do not directly control translation initiation or elongation, then we will con- sider our alternative hypothesis: that a significant fraction of mRNA isoform diversity arises from noisy splicing which are then excluded from polyribosomes by some unknown mRNA surveillance pathway. Solving this important problem will not only reveal how cis-elements influence translational yield, but will also define mechanistic links between the processes of alternative splicing and mRNA translation. In the long-term, our research program will facilitate new opportunities for RNA-based diagnostics and therapies that will be applicable to a wide array of human diseases.