Dissecting the Functions of RNA Helicases in Single Spliceosomes

剖析单剪接体中 RNA 解旋酶的功能

• 批准号: 8830784
• 负责人: Julia Reed Widom
• 金额: $ 5.24万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8830784
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Alternative Splicing; Area; Base Pairing; Biological Models; Biophysics; Boxing; Catalysis; Chemistry; Chicago; Code; Collaborations; Complex; Cystic Fibrosis; Defect; Disease; Dissociation; Dominant-Negative Mutation; Electrophoretic Mobility Shift Assay; Eukaryota; Exons; Family; Fluorescence Resonance Energy Transfer; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Heating; Hereditary Disease; Human; Human Genetics; In Vitro; Introns; Joining Exons; Junk DNA; Kinetics; Knowledge; Letters; Malignant Neoplasms; Measures; Mediating; Mentors; Messenger RNA; Modeling; Molecular; Molecular Conformation; Monitor; Mutation; Nature; Neurodegenerative Disorders; Nucleotides; Open Reading Frames; Organism; Phase; Play; Process; Production; Protein Biosynthesis; Proteins; RNA; RNA Helicase; RNA Splicing; RNA-Protein Interaction; Reaction; Recombinants; Regulation; Relative (related person); Research Personnel; Rest; Role; Small Nuclear Ribonucleoproteins; Solutions; Spectrum Analysis; Spliceosomes; Techniques; Testing; Time; Training; U5 Small Nuclear Ribonucleoprotein; Universities; Work; Writing; Yeasts; fluorophore; helicase; human disease; intein; literature survey; mRNA Precursor; prevent; protein function; public health relevance; research study; single molecule; single-molecule FRET; tool

 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.

 描述（由适用提供）：1970年代，研究人员发现，在许多较高的生物（例如人类）中，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域（“外显子”）分裂，被包含数以万计的核苷酸的“内含子”隔开。我们现在知道，基因组的非蛋白质编码区域编码有助于基因调节，催化等的RNA，并且除去了基因的编码区域的介绍区分开的介绍，并将外显子在称为剪接的过程中连接在一起。基因表达的关键步骤允许精细调节，并通过替代剪接使单个基因编码多种蛋白质。在此过程中的错误可能是致命的 - 据估计，多达60％的人类遗传疾病涉及剪接缺陷。剪接是由剪接体执行的，剪接体是一种多型大分子机器，其功能取决于许多蛋白质和RNA成分之间的相互作用。确定这些成分之间的作用和相互作用对于理解剪接过程至关重要，因此，隐含剪接的许多人类疾病的分子机制。该提案重点介绍了两种蛋白质PRP22和PRP16，它们促进了酵母剪接体中的结构重排（这与人类中的结构非常相似）。这些蛋白质在体外充当RNA解旋酶，但尚不清楚这种解旋酶活性如何有助于它们在剪接体中的功能。具体目标1涉及研究由PRP22诱导的MRNA组成的变化，分别检查其在第二步之后的第二个催化步骤和mRNA产物释放中的作用。特定的目标2着重于PRP16，该PRP16已显示出校对角色，在催化的步骤1之前触发了次优的前MRNA底物的丢弃。该方法将利用单分子荧光共振能传递（SMFRET）的工具，该工具将提供敏感性，不仅比较了剪接中不同中间状态中存在的前MRNA构象，还可以比较其动力学。通过使用PRP22和PRP16中的显性负突变在剪接周期中引入块，将测量这些蛋白质诱导的MRNA构象和动力学的变化。这项工作的一个重要方面是比较PRP16和PRP22对模型底物的解旋酶/ATPase活性，以解决其在剪接体中的活性。该项目提供的知识将与涉及剪接缺陷的疾病的理解和治疗有关。此外，该项目将在RNA生物物理学，单分子光谱以及科学写作，表现和心理方面提供重要的培训。