Mechanism of U1 snRNPs suppression of premature cleavage & polyadenylation

U1 snRNPs抑制过早卵裂的机制

基本信息

批准号：
8802007
负责人：
GIDEON DREYFUSS
金额：
$ 40万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
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/8802007
关键词：
3&apos Untranslated Regions 5&apos Splice Site Address Antisense Oligonucleotides Area Base Pairing Belief Binding Binding Sites Biogenesis Biological Assay Biology Cells Code Complex Deletion Mutation Dimensions Elements Eukaryota FarGo Future Gene Expression Process Gene Expression Profile Gene Expression Regulation Genes Genetic Transcription Genome Goals Introns Knowledge Laboratories Length Life 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 Relative (related person)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 sequencing

项目摘要

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电视脚本的机制，这是我的实验室最近发现的一种新的主要基因表达过程。转录对于真核生物中信使 RNA (mRNA) 合成所必需的大多数蛋白质编码基因的全长 RNA 聚合酶 II (pol II) 转录至关重要。它依赖于 U1 snRNP (U1)（一种丰富的非编码 11 亚基核糖核蛋白颗粒）来保护新生的 pol II 转录物免于因内含子中的切割和多腺苷酸化 (CPA) 提前终止，这种切割和多腺苷酸化 (CPA) 具有高度破坏性。 U1 snRNP 因其在 5' 剪接位点 (ss) 识别中的作用而得到充分表征，这是内含子剪接的关键和第一步。然而，我们发现 U1 具有额外的非剪接功能，作为来自随机存在于大内含子中的神秘多腺苷酸化信号 (PAS) 的过早 CPA (PCPA) 的抑制子。我们将 U1 的 PCPA 抑制也称为转录，因为它对于允许转录走得更远是必要的。相比之下，来自不同 polII 启动子的新生上游反义转录本由于 PAS 与 U1 的结合比例相反而相对不受保护，并且迅速降解，表明转录组在塑造转录组中的一般作用。此外，可以通过 U1 水平的微小变化来精细调节的转录活性也决定了 mRNA 的长度。例如，由于在 3' 非翻译区 (3'UTR) 中使用更近端的 PAS，U1 的轻微减少会导致广泛的缩短，从而消除 mRNA 调节元件，例如抑制翻译的 microRNA 结合位点。基于我们之前的研究，我们提出了一个模型来解释 U1 如何抑制内含子中的 PAS，包括它结合内含子而不仅仅是 5's 的假设。然而，缺乏检验这一假设和理解电视脚本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 U1 抑制的工具，并进行了详细探测的检测。我们建议追求三个具体目标：1）生成活细胞新生转录本上 U1 snRNP 结合位点的全基因组图谱，这将相对于我们最近完成的 PCPA 位点的全基因组图谱进行解释。 2) 确定 U1 snRNP 蛋白和 U1 snRNA 结构域对于电视脚本的作用。 3) 使用我实验室首创的综合 RNP 相互作用组发现方法来识别 CPA 复合体和/或 U1 PCPA 抑制的其他靶标。总之，这些目标将为 U1 snRNP 转录机制提供重要见解，并促进对这一新维度在生物学和医学基因调控方面的理解和潜在应用。