Protein RNA Rearrangements in the Spliceosome

剪接体中蛋白质 RNA 重排

基本信息

批准号：
9311659
负责人：
CHARLES C QUERY
金额：
$ 53.5万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9311659
关键词：
3&apos Splice Site ATP phosphohydrolase Address Base Pairing Binding Binding Sites Bioinformatics Biological Models Biology Catalysis Cell Nucleus Cells ChIP-seq Chemicals Chromatin Code DNA Polymerase II Data Data Set Development Dysmyelopoietic Syndromes Engineering Event Excision Exons Filament Fission Yeast Gene Expression Genes Genetic Transcription Goals Growth Hematopoiesis Human Introns Investigation Joining Exons Knowledge Ligands Link Malignant Neoplasms Mammalian Cell Maps Minor Modernization Modification Mutation Nucleotides Pathway interactions Polyadenylation Positioning Attribute Primer Extension Process Proteins Pseudouridine Publishing RNA RNA Splicing Reaction Repetitive Sequence Role Site Small Nuclear RNA Small Nuclear Ribonucleoproteins Spinal Muscular Atrophy Spliceosome Assembly Pathway Spliceosomes Structure Testing Trinucleotide Repeats U2 Small Nuclear Ribonucleoprotein U2 small nuclear RNA U6 small nuclear RNA Variant Vertebrates Yeasts cell type density differential expression experimental study genetic variant global run on sequencing mRNA Precursor mammalian genome novel programs public health relevance stem transcription factor transcriptome sequencing

项目摘要

ABSTRACT Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how the reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events — which are not understood mechanistically — will elucidate interactions and rearrangements among core components and may serve as a paradigm for rearrangements in the spliceosome and in other RNP machines. This proposal focuses on mechanisms by which spliceosomal dynamics impact splicing fidelity. Experiments will first investigate binding and positioning of the 3'SS-UAG onto the spliceosome. Binding of the spliceosome to the 3'SS is critical for intron definition, for spliceosome assembly, and for splicing catalysis. Yet, nothing is known of spliceosome–3'SS-UAG interaction, other than the early interaction with U2AF. Here we use an `orthogonal spliceosome' (second-copy, reverse-engineered, designer spliceosome) that we have developed in yeast, to identify both the 3'SS binding site for second-step catalysis and a `loading site' for 3'SS on the assembling spliceosome. Second, two large gaps in our understanding of RNA biology are the identification of RNAs between 50 and 200 nts, which are missing in almost all modern-day sequencing datasets, and the bioinformatic analysis of repetitive sequences – the snRNAs represent both. We have identified novel U2 snRNA variants that are expressed differentially in cells, and we will investigate the components, function, and substrates of novel U2-variant spliceosomes.

抽象的通过剪接体从前体信使RNA切除内含子是几乎所有的关键步骤人基因表达。该过程受到高度调节，与基因的转录完全相关，其他加工事件，例如聚腺苷酸化和核苷酸的修饰。剪接体识别化学事件的确切位点的机制以及如何反应是催化的。该项目的长期目标是了解剪接组成分与底物的RNA配体之间的相互作用和重排用于催化反应。多个因素和多个重新排列的大量证据论证分支网站的识别事件。对这些事件的调查 - 尚不理解机械上 - 将阐明核心组件之间的相互作用和重排，可以用作在剪接体和其他RNP机器中重新排列的范例。该提议重点剪接动力学影响剪接保真度的机制。实验将首先研究3'S-UAG在剪接体上的结合和定位。剪接体与3'S的结合对于内含子定义，剪接组装和剪接至关重要催化。然而，除了与早期互动与与 U2AF。在这里，我们使用“正交剪接体”（二次拷贝，反向设计的，设计师剪接体）我们已经在酵母中开发了，以识别用于二步催化的3'S绑定位点和` 在组装剪接体上的3'S站点。第二，我们对RNA生物学的理解中的两个差距是否识别50至200 NT之间的RNA，几乎所有现代测序中都缺少了RNA 数据集以及重复序列的生物信息学分析 - SNRNA代表这两者。我们有鉴定出在细胞中表达不同的新型U2 snRNA变体，我们将研究新型U2变体剪接体的组件，功能和底物。