Understanding the Structural Properties of Triple Helices and a Triple-Stranded RNA-Binding Protein

了解三螺旋和三链 RNA 结合蛋白的结构特性

• 批准号: 10115519
• 负责人: Charlotte Nicole Kunkler
• 金额: $ 4.22万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10115519
• 关键词: 3-Dimensional; Address; Base Pairing; Binding; Binding Proteins; Biochemical; Biological Assay; Cells; Complex; Cryoelectron Microscopy; DNA; DNA Maintenance; Electron Microscopy; Electrophoretic Mobility Shift Assay; Fellowship; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomic DNA; Health; Human; In Vitro; Industry; Insulin-Like Growth-Factor Binding Protein 1; Learning; MALAT1 gene; Major Groove; Malignant Neoplasms; Methods; Methyltransferase; MicroRNAs; Modification; Molecular Structure; Nature; Nucleotides; Organism; Population; Positioning Attribute; Property; Proteins; Purines; Pyrimidine; RNA; RNA Recognition Motif; RNA Stability; RNA-Binding Proteins; Research; Role; Structure; Untranslated RNA; Work; X-Ray Crystallography; Y base; alpha helix; base; career; cryogenics; ds-DNA; gel mobility shift assay; insight; knowledge base; novel; nucleic acid structure; stability testing; three dimensional structure; triple helix

Project Summary Though triple helices were deduced to form in vitro over sixty years ago, the function of triple helices in cells is only beginning to be appreciated. My work will focus on pyrimidine motif triple helices, whereby a pyrimidine- rich third strand binds in the parallel orientation along the purine-rich strand in the major groove of a double helix. This work will focus on two key questions about triple helices: (i) which base triples stabilize an RNA•DNA-DNA triple helix? and (ii) how does a protein recognize a triple helix? Noncoding RNAs have been proposed to regulate gene expression by binding to genomic DNA via an RNA•DNA-DNA triple helix. However, beyond the canonical U•A-T and C•G-C base triples, the stability of base triples that compose RNA•DNA-DNA triple helices is unknown. Therefore, in Aim 1, I will systematically determine the stability of an RNA•DNA-DNA triple helix when a single base triple, Z•X-Y (where Z = C, U, A, G and X-Y = A-T, G-C, T-A, C-G), is varied in a U•A-T-rich triple helix, using a native electrophoretic mobility shift assay to examine the binding between the RNA and double-stranded DNA. Furthermore, I will test the stability of nine common RNA modifications at the same position in the RNA•DNA-DNA triple helix and compare the stabilities of modified RNA to its unmodified RNA counterpart. This study will be the first to show the relative stability of each Z•X-Y base triple in an RNA•DNA-DNA triple helix and will lead to a better understanding of how nature uses RNA•DNA-DNA triple helices to regulate gene expression. In Aim 2, I will use X-ray crystallography and cryogenic electron microscopy to solve a three-dimensional structure of methyltransferase-like protein 16 (METTL16) in complex with the triple helix from the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA. METTL16 is an essential human RNA methyltransferase that has been shown to bind to the 3' end of the MALAT1 long noncoding RNA both in vitro and in cell-based assays. Interestingly, the 3' end of MALAT1 forms a triple helix that functions to protect MALAT1 from 3'-end degradation. METTL16 is the first and only putative triple-stranded RNA-binding protein. Thus, the structure of the METTL16-MALAT1 triple helix complex will potentially uncover a novel class of triple-stranded RNA-binding proteins. Overall, this work will increase our knowledge of base triples that stabilize triple helices and of proteins that interact with triple helices.

项目摘要 虽然三重螺旋在60多年前就被推断为在体外形成，但三重螺旋在细胞中的功能仍然是未知的。 才刚刚开始被欣赏。我的工作将集中在嘧啶基序三螺旋，其中嘧啶- 富含嘌呤的第三条链以平行方向沿着富含嘌呤的链结合在双链的大沟中。 螺旋。这项工作将集中在两个关键问题的三重螺旋：（i）基地三重稳定， RNA·DNA-DNA三螺旋？以及（ii）蛋白质如何识别三螺旋？非编码RNA已经被 提出通过RNA·DNA-DNA三螺旋与基因组DNA结合来调节基因表达。然而，在这方面， 除了典型的U·A-T和C·G-C碱基三联体，组成RNA·DNA-DNA的碱基三联体的稳定性 三重螺旋是未知的。因此，在目标1中，我将系统地确定RNA·DNA-DNA的稳定性。 当单碱基三联体Z·X-Y（其中Z = C、U、A、G和X-Y = A-T、G-C、T-A、C-G）在一个或多个碱基中变化时， 富含U·A-T的三螺旋，使用天然电泳迁移率变动分析来检查 RNA和双链DNA。此外，我将测试9种常见RNA修饰的稳定性， 在RNA·DNA-DNA三螺旋中的相同位置，并比较修饰的RNA与其未修饰的RNA的稳定性 RNA对应物。这项研究将是第一个显示每个Z·X-Y碱基三元组在一个 RNA·DNA-DNA三重螺旋，并将导致更好地了解自然界如何使用RNA·DNA-DNA三重螺旋 螺旋来调节基因表达。在目标2中，我将使用X射线晶体学和低温电子 显微镜下解决了一个三维结构的甲基转移酶样蛋白16（胃L16）在复杂的 与转移相关肺腺癌转录本1（MALAT 1）的三螺旋长 非编码RNA。L16是一种必需的人类RNA甲基转移酶，已被证明与胃蛋白酶结合。 MALAT 1长非编码RNA的3'末端，在体外和基于细胞的测定中。有趣的是， MALAT 1形成三螺旋，其功能是保护MALAT 1免受3 '末端降解。L16是第一个 和仅有的三链RNA结合蛋白。因此，胃L16-MALAT 1三螺旋的结构 复合物将有可能揭示一类新的三链RNA结合蛋白。总的来说，这项工作将 增加了我们对稳定三螺旋的碱基三联体和与三联体相互作用的蛋白质的知识 螺旋