Single Molecule Analysis of Spliceosome Catalysis and Fidelity

描述(申请人提供)：前信使核糖核酸剪接是真核基因表达的重要步骤。内含子切除和外显子连接的过程是由被称为剪接体的RNA和蛋白质的巨型道尔顿复合体执行的。这一建议旨在开发和利用新的化学和生物物理方法在单分子水平上研究前mRNA剪接的机制。在指导阶段，将发展位置特异性荧光标记前mRNAs和酵母剪接体蛋白的方法，以便在单分子荧光显微镜下观察它们在完整剪接反应中的动态。这项工作将集中于解决与剪接体在能够执行套索形成的酶和能够连接外显子的酶之间的转换相关的问题，包括直接观察DExD/H-box蛋白/保真度因子如Prp16和Prp22与剪接体的相互作用。在独立阶段，将向人类剪接体过渡。这项工作将集中在人类剪接特有的问题上，例如SR蛋白在剪接的化学步骤中对前mRNA动态的影响。此外，还将开发和使用化学方法来提供3‘剪接位点识别和外显子连接的高动力学分辨率分析，使用光笼基团和光在溶液和单分子水平上触发这些事件。作为麻省理工学院JoAnne Stubbe的研究生，我在机械酶学和核苷酸合成方面接受了广泛的培训，同时学习从头开始的嘌呤生物合成。因此，在我独立的职业生涯中，我希望使用化学和详细的动力学方法来研究复杂的细胞机，如剪接体。作为NIH NRSA与梅丽莎·摩尔和杰夫·盖尔斯的博士后研究员，我扩大了我的培训范围，包括RNA生物化学、酵母遗传学和单分子荧光显微镜。这种环境使我能够制定一条职业道路，在这条道路上，我将能够利用化学和物理学对人类生物学提供独特和基本的见解。 相关性：前信使核糖核酸剪接是人类基因表达的一个重要步骤，因此对人类健康和生物学的研究具有重要意义。绝大多数人类基因是交替剪接的，这一过程中的缺陷可能占人类遗传病的15%。这里提出的研究将通过阐明基因表达的这一步骤对人类健康产生重大影响。

DESCRIPTION (provided by applicant): Pre-mRNA splicing is an essential step in eukaryotic gene expression. The processes of intron excision and exon ligation are carried out by a mega-Dalton complex of RNAs and proteins called the spliceosome. This proposal seeks to develop and utilize new chemical and biophysical methods to study the mechanism of pre-mRNA splicing at the single molecule level. During the mentored phase, methods will be developed to site-specifically fluorescently label pre-mRNAs and yeast spliceosomal proteins in order to observe their dynamics during a complete splicing reaction by single molecule fluorescence microscopy. This work will focus on addressing questions relevent to the transition of the spliceosome between an enzyme capable of carrying out lariat formation and an enzyme competent for exon ligation, including direct observation of the interaction of DExD/H-box proteins/fidelity factors such as Prp16 and Prp22 with the spliceosome. During the independent phase, a transition will be made to the human spliceosome. This work will focus on questions specific to human splicing such as the influence of SR proteins on pre-mRNA dynamics during the chemical steps of splicing. In addition, chemical methods will be developed and employed to provide a high kinetic resolution analysis of 3' splice site recognition and exon ligation using photocaging groups and light to trigger these events in solution and at the single molecule level. I have received extensive training in mechanistic enzymology and nucleotide synthesis while studying de novo purine biosynthesis as a graduate student with JoAnne Stubbe at MIT. As a result, I wish to employ chemical and detailed kinetic methods to the study of complex cellular machines such as the spliceosome during my independent career. As a NIH NRSA postdoctoral research fellow with Melissa Moore and Jeff Gelles, I have expanded my training to include RNA biochemistry, yeast genetics, and single molecule fluorescence microscopy. This enivronment has allowed me to formulate a career path in which I will be able to use chemistry and phyiscs to provide unique and fundamental insight into human biology. RELEVANCE: Pre-mRNA splicing is an essential step in human gene expression and is therefore of fundamental importance to the study of human health and biology. The vast majority of human genes are alternatively spliced and defects in this process may account for >15% of human genetic diseases. The research proposed here will have a significant impact on human health by elucidating this step in gene expression.