Structural and dynamics studies of post-transcriptionally modified snRNAs

项目摘要： 越来越多的证据表明，转录后修饰在生物学过程中起着重要作用。 编码和非编码RNA的功能。已经有超过100种化学修饰的RNA核苷被发现。 迄今为止发现的可以影响RNA命运和功能的基因。2 '-OH位置的2'-O-甲基化（Nm）是一种 影响所有四个核苷的核糖部分的独特修饰。Nm在高 核糖体RNA（rRNA）、转移RNA（tRNA）和小核RNA（snRNA）丰度。它也存在 在microRNA上，在信使RNA（mRNA）的5 '-帽上，最近在mRNA内部发现。Nm是 对于上述许多RNA的正常功能至关重要，在某些情况下，Nm的丢失与 临床条件。尽管它很重要，但Nm如何影响RNA细胞活性仍然知之甚少 在分子水平上对这些RNA的大部分。许多调节RNA的细胞功能依赖于 RNA结构动力学的微调变化，这些变化是响应特定的细胞信号而发生的， 结合蛋白、配体或其他RNA。一个突出的例子是剪接体机制， 催化mRNA成熟。RNA动力学在RNA的组装和拆卸中起着重要的作用。 剪接体以及催化所需的不同构象状态之间的循环。U2-U6和 U4-U6 snRNA复合物是剪接体的关键动态结构元件，其高度依赖于剪接体。 富含Nm修饰。这些修饰对snRNA的作用仍然知之甚少。这 该项目将确定Nm修饰如何影响snRNA结构和剪接。目标1和2将利用 先进的核磁共振（NMR）技术，包括弛豫色散实验（RD）， 和另外的生物物理技术来测试Nm修饰的损失影响稳定性的假设， 杂交动力学和snRNA结构的构象动力学。目标3将使用siRNA介导的 敲除和遗传定义的敲除细胞系，以确定哪些snRNA修饰具有 对心肌细胞中mRNA剪接的影响最大。目标1和2的结构动力学研究将 因此，补充了目标3的功能研究。总之，这些研究将大大扩大我们的 了解Nm修饰如何通过改变基因的表达来促进活性（例如心脏基因的剪接） snRNA的动力学和结构特性。

Project Summary: There is increasing evidence that post-transcriptional modifications play essential roles in the biological functions of coding and non-coding RNAs. More than 100 chemically-modified RNA nucleosides have been identified to date that can impact RNA fate and function. 2'-O-methylation (Nm) of the 2'-OH position is a unique modification that impacts the ribose sugar moiety of all four nucleosides. Nm is found in high abundance in ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA). It is also present on microRNA, at the 5'-cap of messenger RNA (mRNA), and recently discovered internally on mRNA. Nm is critical for the proper functioning of many of the above RNAs, and in several cases, loss of Nm has been linked to clinical conditions. Despite its importance, how Nm affects RNA cellular activity remains poorly understood at the molecular level for the majority of these RNAs. The cellular functions of many regulatory RNAs rely on finely-tuned changes in RNA structural dynamics that take place in response to specific cellular cues such as the binding proteins, ligands, or other RNAs. A prominent example is the spliceosome machinery, which catalyzes mRNA maturation. RNA dynamics plays essential roles in the assembly and disassembly of the spliceosome as well as in cycling between the different conformational states required for catalysis. U2-U6 and U4-U6 snRNA complexes are critical dynamic structural elements of the spliceosome, which are highly enriched in Nm modifications. The role of these modifications on snRNAs remains poorly understood. This project will determine how Nm modifications influence snRNA structure and splicing. Aims 1 and 2 will utilize advanced Nuclear Magnetic Resonance (NMR) techniques, including Relaxation Dispersion experiments (RD), and additional biophysical techniques to test the hypothesis that loss of Nm modifications affects the stability, hybridization kinetics, and conformational dynamics of snRNA structures. Aim 3 will use siRNA-mediated knockdown and genetically-defined knockout cell lines to determine which snRNA modifications have the greatest impact on mRNA splicing in cardiac cells. The structural dynamics studies of Aims 1 and 2 will therefore complement the functional studies of Aim 3. Together, these studies will significantly expand our knowledge of how Nm modifications contribute to activity (for instance splicing of cardiac genes) via altering the dynamic and structural properties of snRNAs.