Biophysical investigations of RNA complexes essential for gene expression

项目摘要 拟议的研究重点是应用生物物理学方法来了解的结构和功能， 真核细胞中调节基因表达的RNA-蛋白质复合物。我们的调查将阐明 剪接体组装途径中未探索的步骤，mRNA衰变和RNA沉默。我们建议 确定U6小核核糖核蛋白（U6 snRNP）颗粒的结构，并描述它如何 与U4 snRNP缔合以形成U4/U6 di-snRNP。这些相互作用对于组装和 剪接体的再循环，但尚未在分子水平上理解。我们还将研究LSM1- 7复合物，其与U6 snRNP蛋白高度相关，但指导mRNA衰变，这是一个主要的转录后 基因表达稳态水平的决定因素。我们最近的数据表明Lsm 1 - 7复合物是 由其辅因子Pat1重塑，以便与广泛的mRNA结合。最后，我们将探索一种新的 发现了RNA生物学的一个新领域，涉及酶促将聚UG（pUG）尾加到RNA的3 ′末端。pug- 加尾RNA是基因沉默的有效试剂，并建立了反式所需的分子记忆。 线虫的世代表观遗传。我们发现pUG RNA折叠成一种不寻常的 四链体结构，解释了体内RNA沉默的长度要求。人类有超过 神经元内含子内的数千个内部pUG序列，其用于调节选择性前体mRNA剪接 通过与TDP-43蛋白的相互作用。我们已经发现pUG RNA保持其四链体， 当与TDP-43结合时，其结构，后者参与相变和神经退行性变 疾病我们将研究这种相互作用的结构基础，并将在以下方面合作检验我们的假设： 动物通过阐明pUG RNA如何与蛋白质和小分子结合， 这些相互作用如何在不同的途径中指导不同的生物学结果，如RNA沉默， 选择性剪接

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.