Dissecting the Functions of RNA Helicases in Single Spliceosomes

 描述(申请人提供)：一项令人惊讶的发现是在20世纪70年代，研究人员发现，在许多高等生物，如人类，只有一小部分基因组编码蛋白质，而其余的似乎是“垃圾DNA”。此外，绝大多数基因的蛋白质编码区(“外显子”)被包含多达数万个核苷酸的“内含子”分开。我们现在知道，基因组的非蛋白质编码区编码有助于基因调节、催化等的RNA，分离基因编码区的内含子被移除，外显子在一个称为剪接的过程中连接在一起。基因表达的这一关键步骤允许进行精细的调控，并通过选择性剪接允许单个基因编码不止一种蛋白质。这一过程中的错误可能是致命的-据估计，高达60%的人类遗传病涉及剪接缺陷。剪接是由剪接体执行的，剪接体是一个几百万吨的大分子机器，其功能依赖于许多蛋白质和RNA成分之间的相互作用。确定这些组分的作用和相互作用对于理解剪接过程以及许多与剪接有关的人类疾病的分子机制至关重要。这项建议侧重于两种蛋白质，Prp22和Prp16，它们促进酵母剪接体(与在人类中发现的非常相似)的结构重排。这些蛋白质在体外作为RNA解旋酶发挥作用，但目前尚不清楚这种解旋酶活性如何影响它们在剪接体中的功能。具体目标1包括研究Prp22诱导的前mRNA构象的变化，分别研究Prp22在第二步剪接催化步骤中的作用以及在第二步之后对mRNA产物释放的作用。特定目的2专注于Prp16，它已被证明起到校对作用，在催化步骤1之前触发次优的前mRNA底物的丢弃。该方法将利用单分子荧光共振能量转移(SmFRET)工具，该工具将提供灵敏度，不仅可以比较剪接中不同中间状态下存在的前mRNA构象，而且还可以比较它们的动力学。通过在Prp22和Prp16中使用显性负突变在剪接循环中引入块，将测量由这些蛋白质诱导的前mRNA构象和动力学的变化。这项工作的一个重要方面将是比较Prp16和Prp22在溶液中模型底物上的解旋酶/ATPase活性与它们在剪接体中的活性。该项目提供的知识将与理解和治疗涉及剪接缺陷的疾病有关。此外，该项目将在RNA生物物理学、单分子光谱学以及科学写作、演讲和指导方面提供重要的培训。

 DESCRIPTION (provided by applicant): A surprising discovery was made in the 1970s when researchers found that in many higher organisms, such as humans, only a small fraction of the genome encodes proteins, whereas the rest is seemingly "junk DNA". In addition, the vast majority of genes have their protein-coding regions ("exons") split up, separated by "introns" containing up to tens of thousands of nucleotides. We now know that non-protein-coding regions of the genome encode RNAs that contribute to gene regulation, catalysis and more, and the introns separating coding regions of genes are removed and the exons are joined together in a process called splicing. This critical step in gene expression allows for exquisitely fine-tund regulation and, through alternative splicing, allows a single gene to encode for more than one protein. Mistakes in this process can be lethal - it has been estimated that up to 60% of human genetic diseases involve defects in splicing. Splicing is executed by the spliceosome, a multi-megaDalton macromolecular machine whose function depends on the interplay between many protein and RNA components. Determining the roles of and interactions between these components is of central importance to understanding the process of splicing and, therefore, the molecular mechanisms of the many human diseases in which splicing is implicated. This proposal focuses on two proteins, Prp22 and Prp16, which facilitate structural rearrangements in the yeast spliceosome (which is very similar to that found in humans). These proteins function as RNA helicases in vitro, but it is not known how this helicase activity contributes to their function in the spliceosome. Specific Aim 1 involves studying the changes to pre-mRNA conformation induced by Prp22, separately examining its roles in the second catalytic step of splicing and in mRNA product release after the second step. Specific Aim 2 focuses on Prp16, which has been shown to play a proofreading role, triggering the discard of suboptimal pre- mRNA substrates prior to step 1 of catalysis. The approach will utilize the tools of single-molecule fluorescence resonance energy transfer (smFRET), which will provide the sensitivity to compare not only the pre-mRNA conformations present in different intermediate states in splicing, but also their dynamics. By introducing blocks in the splicing cycle using dominant negative mutations in Prp22 and Prp16, the changes in pre-mRNA conformation and dynamics induced by these proteins will be measured. An important facet of this work will be comparing the helicase/ATPase activities of Prp16 and Prp22 on model substrates in solution to their activities in the spliceosome. The knowledge provided by this project will be relevant to the understanding and treatment of diseases that involve defects in splicing. In addition, the project will provide important training in the areas of RNA biophysics, single-molecule spectroscopy, and scientific writing, presentation and mentoring.