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中产生mRNA多样性的一种强大机制。来自同一基因的不同的信使核糖核酸异构体可能彼此之间有细微的差异，或者在它们的序列组成上有根本的变化。值得注意的是，人们对这种不同的信使核糖核酸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之前的研究，即选择性剪接的过程影响所产生的mRNA异构体的翻译效率。在这项建议中，我们使用全基因组和分子方法来解开错综复杂的转录后基因表达耦合机制。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患病率和影响。在这个目标中，我们将使用我们最近开发的Frac-Seq方法对15个不同的ENCODE细胞系和两个不同的灵长类干细胞模型系统中翻译的多聚核糖体的mRNA异构体进行量化。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SRSF1为模型，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，我们将使用CLIP-Seq定位SRSF1或非穿梭突变体在不同亚细胞组分中的结合位点；使用Frac-Seq确定AS-TC对SRSF1的依赖性；使用RNA亲和层析发现新的反式AS-TC因子。这项工作的结果将为穿梭蛋白(如SRSF1)在转录后基因表达协调中的功能(S)定义一个新的范式。(3)确定调控异构体特异性mRNA翻译的分子机制。为此，我们将使用FragSeq和Quepasa阐明调节多聚核糖体结合的全部RNA元件和结构，然后从生化角度确定AS-TC中涉及的序列元件如何调节翻译起始和延伸机制。通过确定AS-TC元件如何调控mRNA翻译，我们将能够驳斥或支持这一假设的中心假设，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能直接控制翻译的起始或延伸，那么我们将 考虑到我们的另一种假设：有相当一部分的信使核糖核酸异构体多样性 从嘈杂的剪接，然后通过一些未知的mRNA监测途径排除在多聚核糖体中。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，而且还将确定选择性剪接过程与mRNA翻译之间的机制联系。从长远来看，我们的研究计划将促进基于RNA的诊断和治疗的新机会，这些诊断和治疗将适用于广泛的人类疾病。

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.