Mechanism of U1 snRNPs suppression of premature cleavage & polyadenylation

U1 snRNPs抑制过早卵裂的机制

• 批准号: 9179656
• 负责人: GIDEON DREYFUSS
• 金额: $ 40万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-10 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9179656
• 关键词: 3' Untranslated Regions; 5' Splice Site; Address; Antisense Oligonucleotides; Area; Base Pairing; Belief; Binding; Binding Sites; Biogenesis; Biological Assay; Biology; Cells; Code; Complex; DNA Polymerase II; DNA-Directed RNA Polymerase; Deletion Mutation; Dimensions; ENG gene; Elements; Eukaryota; Future; Gene Expression Process; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Informatics; Introns; Knowledge; Laboratories; Length; Maps; Mass Spectrum Analysis; Medicine; Messenger RNA; Methodology; MicroRNAs; Modeling; Nuclear RNA; Organism; Play; Polyadenylation; Polymerase; Positioning Attribute; Process; Protein Isoforms; Proteins; RNA; RNA Polymerase II; RNA Splicing; RNA chemical synthesis; RNA, Messenger, Splicing; RNA-Binding Proteins; Ribonucleoproteins; Role; Shapes; Signal Transduction; Site; Structure; Testing; Textbooks; Therapeutic; Transcript; Translations; U1 Small Nuclear Ribonucleoprotein; U1 small nuclear RNA; Untranslated RNA; base; crosslink; genome-wide; insight; knock-down; particle; premature; promoter; public health relevance; tool; transcriptome; transcriptome sequencing

DESCRIPTION (provided by applicant): This proposal's overarching goal is to understand the mechanism of telescripting, a new and major gene expression process recently discovered in my laboratory. Telescripting is essential for full length RNA polymerase II (pol II) transcription from the majority of protein-coding genes in eukaryotes necessary for messenger RNA (mRNA) synthesis. It relies on U1 snRNP (U1), an abundant non-coding 11-subunit ribonucleoprotein particle, to protect nascent pol II transcripts from early termination by cleavage and polyadenylation (CPA) in introns, which is highly destructive. U1 snRNP is well characterized for its role in 5' splices site (ss) recognition, a key and the first step in splicing of introns. Howeer, we found that U1 has an additional non-splicing function as a suppressor of premature CPA (PCPA) from cryptic polyadenylation signals (PASs) that are stochastically present in large introns. We refer to U1's PCPA suppression also as telescripting, as it is necessary for allowing transcription to go farther. In contrast, nascent upstream antisense transcripts from divergent polII promoters are relatively unprotected due to an inverse PAS to U1 binding ratio and are rapidly degraded, indicating telescripting's general role in shaping the transcriptome. Furthermore, telescripting activity, which can be finely modulated by slight changes in U1 level also determines mRNA length. For example, slight U1 decrease causes widespread shortening due to usage of more proximal PASs in the 3' untranslated region (3'UTR), thereby removing mRNA-regulating elements such as translation repressing microRNA binding sites. Based on our previous studies we proposed a model to explain how U1 suppresses PASs in introns, including the hypothesis that it binds in introns and not only to 5'ss. However, basic information needed to test this hypothesis and for understanding telescripting mechanism is lacking. To address this, my laboratory established tools to precisely manipulate PCPA and its suppression by U1, and assays to probe it in detail. We propose to pursue three specific aims: 1) To generate genome wide map of U1 snRNP binding sites on nascent transcripts in live cells, which will be interpreted relative to genome wide map of PCPA sites that we recently completed. 2) To determine the role of U1 snRNP proteins and essential U1 snRNA domain(s) for telescripting. 3) To identify CPA complex and/or other targets of U1's PCPA suppression using a comprehensive RNP interactome discovery approach pioneered in my laboratory. Together, these aims will provide important insights into mechanism of U1 snRNP telescripting, and advance understanding and potential applications of this new dimension in gene regulation to biology and medicine.

描述（由申请人提供）：该提案的总体目标是了解伸缩的机制，这是我最近在我的实验室中发现的一个新的主要基因表达过程。伸缩伸缩对于从大多数蛋白质编码基因的真核生物中所需的Messenger RNA（mRNA）合成所需的大多数蛋白质编码基因的转录至关重要。它依赖于U1 SNRNP（U1），这是一种丰富的非编码11-亚基核糖核蛋白颗粒，以保护新生的Pol II转录本在内含子中通过裂解和多腺苷酸化（CPA）的早期终止，这是高度破坏性的。 U1 SNRNP在5'接头位点（SS）识别中的作用，键和内含子剪接的第一步。 Howeer，我们发现U1具有额外的非切换函数，作为来自隐性聚腺苷酸化信号（PASS）的过早CPA（PCPA）的抑制，它们在大型内含子中随机存在。我们也将U1的PCPA抑制作用称为伸缩，因为允许转录走得更远的必要条件。相比之下，由于PAS与U1结合率的反相反，来自发散性polii启动子的新生上游上游反义转录物相对不受保护，并且迅速降解，表明伸缩伸缩在塑造转录组中的一般作用。此外，伸缩活性可以通过U1水平的轻微变化来细致调节的伸缩活性也决定了mRNA长度。例如，由于在3'未翻译区域（3'UTR）中使用更多近端通行证，因此轻微的U1减少会导致广泛的缩短，从而消除了mRNA调节的元素，例如翻译抑制MicroRNA结合位点。基于我们以前的研究，我们提出了一个模型，以解释U1如何抑制内含子的通过，包括它在内含子中结合的假设，不仅与5's结合。但是，缺乏检验这一假设和理解伸缩机制所需的基本信息。为了解决这个问题，我的实验室建立了精确操纵PCPA及其抑制U1的工具，并对其进行详细探查。我们建议追求三个具体目标：1）在活细胞中的新生转录本上生成U1 SNRNP结合位点的基因组宽图，这些图将相对于我们最近完成的PCPA位点的基因组广泛图来解释。 2）确定U1 SNRNP蛋白和必需的U1 snRNA结构域的作用。 3）使用在我的实验室中率先使用的全面RNP Intervisome发现方法来识别U1 PCPA抑制的CPA复合物和/或其他靶标。这些目标将共同提供对U1 SNRNP伸缩机制的重要见解，并提高这种新维度在生物学和医学中的新维度的理解和潜在应用。