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

项目摘要 虽然三螺旋在60多年前就在体外形成，但三螺旋在细胞中的功能是 才开始被赏识。我的工作将集中在嘧啶基序的三螺旋，从而一个嘧啶- 在双链的主槽中，富第三链沿富嘌呤链平行结合。 螺旋。这项工作将集中在关于三螺旋的两个关键问题上：(I)哪些碱基三螺旋稳定了一个 RNA·DNA-DNA三螺旋？以及(Ii)蛋白质如何识别三螺旋结构？非编码RNA已经被 提出通过RNA·DNA-DNA三螺旋与基因组DNA结合来调控基因表达。然而， 除了规范的U·A-T和C·G-C碱基三元组外，组成RNA·DNA-DNA的碱基三元组的稳定性 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 当单个碱基三元组Z·X-Y(式中Z=C、U、A、G和X-Y=A-T、G-C、T-A、C-G)在 富含U·A-T的三螺旋，使用天然的电泳迁移率改变分析来检测 核糖核酸和双链DNA。此外，我将测试九种常见的RNA修饰的稳定性 在RNA·DNA-DNA三螺旋中的相同位置，并比较修饰前后的RNA的稳定性 RNA的对应物。这项研究将第一次展示每个Z·X-Y碱基三元组在一个 RNA·DNA-DNA三螺旋结构，将有助于更好地理解自然界如何利用RNA·DNA-DNA三螺旋结构 螺旋来调节基因的表达。在目标2中，我将使用X射线结晶学和低温电子 显微技术解析甲基转移酶样蛋白16(METTL16)复合体的三维结构 与转移相关的肺腺癌转录本1(MALAT1)长的三螺旋 非编码RNA。METTL16是一种重要的人类RNA甲基转移酶，已被证明与 MALAT1长的非编码RNA的3‘端在体外和基于细胞的检测中都是如此。有趣的是，3‘端的 MALAT1形成一个三螺旋结构，起到保护MALAT1免受3‘端降解的作用。METTL16是第一个 和假定的三链RNA结合蛋白。因此，METTL16-MALAT1三螺旋的结构 该复合体可能会发现一类新的三链RNA结合蛋白。总的来说，这项工作将 增加我们对稳定三螺旋的碱基三螺旋和与三螺旋相互作用的蛋白质的知识 螺旋。

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.