Biophysical investigations of RNA complexes essential for gene expression

基因表达必需的 RNA 复合物的生物物理学研究

基本信息

批准号：
10410512
负责人：
Samuel E Butcher
金额：
$ 50.52万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-15 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10410512
关键词：
Alternative Splicing Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Animals Area Binding Biological Biology Biophysics Cells Complex Congenital Abnormality Data Defect Dementia Developmental Delay Disorders Essential Genes Eukaryotic Cell Gene Expression Gene Proteins Gene Silencing Genetic Diseases Genetic Transcription Human Introns Investigation Length Lobe Memory Messenger RNA Molecular Mutation Nematoda Neurodegenerative Disorders Neurons Neutropenia Outcome Pathway interactions Phase Transition Proteins RNA RNA Folding RNA Interference RNA Splicing RNA-Binding Proteins RNA-Protein Interaction Recycling Research Small Nuclear Ribonucleoproteins Spliceosome Assembly Pathway Spliceosomes Structure System Tail Testing U4 Small Nuclear Ribonucleoproteins U6 Small Nuclear Ribonucleoproteins biophysical techniques cofactor in vivo mRNA Decay mRNA Precursor protein TDP-43 protein complex small molecule transgenerational epigenetic inheritance

项目摘要

Project Summary The proposed research is focused on applying biophysical methods to understand the structure and function of RNA-protein complexes that regulate gene expression in eukaryotic cells. Our investigations will elucidate unexplored steps in the spliceosome assembly pathway, mRNA decay and RNA silencing. We propose to determine the structure of the U6 small nuclear ribonucleoprotein (U6 snRNP) particle and to describe how it associates with the U4 snRNP to form the U4/U6 di-snRNP. These interactions are critical for assembly and recycling of the spliceosome but are not yet understood at the molecular level. We will also investigate the Lsm1- 7 complex, which is highly related to U6 snRNP proteins but directs mRNA decay, a major post-transcriptional determinant of steady-state levels of gene expression. Our recent data suggest the Lsm1-7 complex is remodeled by its cofactor Pat1 in order to bind to a broad range of mRNAs. Finally, we will explore a newly discovered area of RNA biology involving the enzymatic addition of poly-UG (pUG) tails to RNA 3¢ ends. pUG- tailed RNAs are potent agents of gene silencing and establish the molecular memories required for trans- generational epigenetic inheritance in nematodes. We have discovered that pUG RNAs fold into an unusual quadruplex structure that explains the length requirement for RNA silencing in vivo. Humans have over a thousand internal pUG sequences within neuronal introns, which serve to regulate alternative pre-mRNA splicing through interactions with the protein TDP-43. We have discovered that pUG RNAs maintain their quadruplex structure when bound to TDP-43, the latter of which is involved in phase transitions and neurodegenerative disease. We will investigate the structural basis for this interaction and will collaboratively test our hypothesis in animals. By elucidating how pUG RNAs associate with proteins and small molecules we will better understand how these interactions direct distinct biological outcomes in diverse pathways such as RNA silencing and alternative splicing.

项目概要拟议的研究重点是应用生物物理学方法来了解我们的研究将阐明调节真核细胞基因表达的 RNA-蛋白质复合物。我们建议剪接体组装途径、mRNA 衰减和 RNA 沉默中未探索的步骤。确定 U6 小核核糖核蛋白 (U6 snRNP) 颗粒的结构并描述其如何与 U4 snRNP 结合形成 U4/U6 di-snRNP 这些相互作用对于组装和形成至关重要。剪接体的回收，但尚未在分子水平上得到了解，我们还将研究 Lsm1-。 7 复合体，与 U6 snRNP 蛋白高度相关，但指导 mRNA 衰减，这是一种主要的转录后过程我们最近的数据表明 Lsm1-7 复合物是基因表达稳态水平的决定因素。通过其辅因子 Pat1 进行重塑，以便与广泛的 mRNA 结合。发现了 RNA 生物学领域，涉及将聚 UG (pUG) 尾部酶促添加到 RNA 3￠末端。带尾的 RNA 是基因沉默的有效媒介，并建立反式转录所需的分子记忆。我们发现 pUG RNA 折叠成一种不寻常的线虫世代表观遗传。四链体结构解释了人类体内 RNA 沉默的长度要求。神经元内含子内有数千个内部 pUG 序列，用于调节选择性前 mRNA 剪接通过与蛋白质 TDP-43 的相互作用，我们发现 pUG RNA 保持其四链体。与 TDP-43 结合时的结构，后者参与相变和神经退行性变我们将研究这种相互作用的结构基础，并共同检验我们的假设。通过阐明 pUG RNA 如何与蛋白质和小分子结合，我们将更好地理解。这些相互作用如何在不同的途径中产生不同的生物学结果，例如RNA沉默和选择性拼接。