Regulation of mRNA isoform fate

mRNA 同种型命运的调控

• 批准号: 9275675
• 负责人: Jeremy Robert Sanford
• 金额: $ 4.54万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-05 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9275675
• 关键词: Affinity Chromatography; Alternative Splicing; Arginine; Attenuated; Binding; Binding Sites; Biological; Biological Models; Cell Line; Cell model; Cells; Code; Collection; Complex; Coupled; Coupling; Cystic Fibrosis; Data; Dependency; Diabetes Mellitus; Diagnostic; Elements; Enhancers; Event; Gene Expression; Gene Expression Regulation; Genes; Genome; Goals; Health; Human; Link; Malignant Neoplasms; Maps; Messenger RNA; Methodology; Methods; Modeling; Molecular; Nonsense-Mediated Decay; Output; Pathway interactions; Polyribosomes; Prevalence; Primates; Problem Solving; Process; Property; Protein Biosynthesis; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Processing; RNA Splicing; RNA, Messenger, Splicing; RNA-Binding Proteins; Regulation; Regulatory Pathway; Research; Ribonucleoproteins; Role; Serine; Spinal Muscular Atrophy; Stem cells; Structure; Subcellular Fractions; Testing; Trans-Activators; Translating; Translation Initiation; Translations; Work; base; cis acting element; crosslinking and immunoprecipitation sequencing; genome-wide; human disease; improved; mutant; novel; particle; programs; research study; transcriptome

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.

描述（由申请人提供）： 替代剪接的过程（AS）是从蛋白质编码基因产生mRNA多样性的强大机制。来自同一基因的替代mRNA同工型可以彼此微妙地不同，也可以在其序列组成中具有根本变化。值得注意的是，关于这种不同的mRNA库是否转化为蛋白质，知之甚少。解决此问题的一个有趣的线索来自我们以前的研究表明，替代剪接过程会影响所得mRNA同工型的转化效率。在此提案中，我们使用全基因组和分子方法来解开复杂的机制在转录基因表达后耦合。为了实现这一目标，我们提出以下特定目的：（1）确定AD-TC在人类基因调节中的患病率和影响。在此目标中，我们将使用我们最近开发的FRAC-SEQ方法来量化15种不同编码细胞系中的翻译多核糖体和两个不同的灵长类动物干细胞模型系统中的mRNA同工型。这些实验将揭示AS-TC调节事件的范围，并确定这是否是用于基因表达的空间或时间调节的保守机制。 （2）使用丝氨酸和精氨酸富蛋白SRSF1作为模型，我们将阐明将RNA结合蛋白在转录后基因调节偶联中的作用和靶标。在此目的中，我们将使用夹子seq绘制不同亚细胞级分中SRSF1或非裂痕突变体的结合位点；使用FRAC-SEQ确定AS-TC对SRSF1的依赖性；使用RNA亲和力色谱法发现新型的跨作用AS-TC因子。这项工作的结果将定义一个新的范式，用于在转录后基因表达的协调中，诸如SRSF1之类的穿梭蛋白的功能。 （3）确定调节同工型特异性mRNA翻译的分子机制。在此目标中，我们将阐明使用fragseq和quepasa调节多核糖体关联的RNA元素和结构的完整曲目，然后确定生物化学上涉及AS-TC涉及的序列元素如何调节翻译启动和伸长的机制。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该提案的中心假设，即替代剪接会产生具有不同转化效率的同工型。如果与差分多核糖体关联相关的序列不能直接控制翻译起始或伸长，那么我们将 考虑我们的替代假设：出现很大一部分mRNA同工型多样性 从嘈杂的剪接中，这些拼接被一些未知的mRNA监视途径排除在多核糖体之外。解决这个重要的问题不仅会揭示顺式元素如何影响翻译产量，还将定义替代剪接和mRNA翻译过程之间的机理联系。从长远来看，我们的研究计划将为基于RNA的诊断和疗法提供新的机会，这些诊断和疗法将适用于各种各样的人类疾病。