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

• 批准号: 10181870
• 负责人: Samuel E Butcher
• 金额: $ 57.47万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 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/10181870
• 关键词: Alternative Splicing; Alzheimer' 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

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.

项目摘要 拟议的研究重点是应用生物物理方法来了解 调节真核细胞基因表达的RNA蛋白质复合物。我们的投资将阐明 在剪接体组装途径，mRNA衰减和RNA沉默中未开发的步骤。我们建议 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相关联，形成U4/U6 DI-SNRNP。这些相互作用对于集会至关重要 剪接体的回收利用，但尚未在分子水平上理解。我们还将研究LSM1- 7复合物，与U6 SNRNP蛋白高度相关，但指导mRNA衰变，这是主要的转录后。 基因表达稳态水平的决定因素。我们最近的数据表明LSM1-7复合物是 通过其辅助因子PAT1进行重塑，以与广泛的mRNA结合。最后，我们将探索一个新的 发现RNA生物学区域，涉及将聚-UG（PUG）尾巴添加到RNA 3¢末端的区域。 p 尾rNA是基因沉默的潜在药物，并建立了反式的分子记忆 线虫中的世代表观遗传。我们已经发现哈巴狗rnas折叠成一个不寻常的 四链体结构解释了体内RNA沉默的长度要求。人类有一个 神经元内含子中的千次内部哈巴狗序列，这些序列用于调节替代性mRNA剪接 通过与蛋白质TDP-43的相互作用。我们发现Pug RNA保持四边形 结构与TDP-43结合时，后者参与相变和神经退行性 疾病。我们将研究这种互动的结构性基础，并将协作检验我们的假设 动物。通过阐明PUG RNA如何与蛋白质和小分子相关联，我们将更好地理解 这些相互作用如何在RNA沉默和 替代剪接。