Biophysical investigations of RNA complexes essential for gene expression

项目摘要 建议的研究集中在应用生物物理方法来了解细胞的结构和功能。 调节真核细胞中基因表达的RNA-蛋白质复合体。我们的调查将澄清 剪接体组装途径、mRNA衰变和RNA沉默中的未知步骤。我们建议 确定U6小核糖核蛋白(U6 SnRNP)颗粒的结构并描述它是如何 与U4 snRNP结合形成U4/U6 di-SnRNP。这些相互作用对于组装和 剪接体的再循环，但在分子水平上还不清楚。我们还将调查LSM1- 7复合体，它与U6 SnRNP蛋白高度相关，但指导mRNA的衰退，这是一种主要的转录后转录 基因表达稳定水平的决定因素。我们最近的数据表明Lsm1-7复合体 被其辅因子Pat1重塑，以便与广泛的mRNAs结合。最后，我们将探索一种新的 已发现的RNA生物学区域，涉及将聚UG(PUG)尾巴酶加到RNA3+末端。帕格- 拖尾RNA是基因沉默的有效试剂，并建立反式转录所需的分子记忆。 线虫的世代表观遗传。我们已经发现巴格RNA折叠成一个不同寻常的 四链结构，解释了体内RNA沉默的长度要求。人类有一个以上的 神经元内含子中的数千个内部PUG序列，用于调节备选的Pre-mRNA剪接 通过与蛋白质TDP-43的相互作用。我们已经发现巴格RNA保持着它们的四链结构 结构与TDP-43结合，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 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不同的途径中引导不同的生物结果，如RNA沉默和 另一种拼接。

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.