Pre-mRNA Splicing Mechanisms

描述（由申请人提供）：该项目的长期目标是阐明通过pre-mRNA剪接过程从新生RNA转录物中去除中间序列或内含子的详细分子机制。真核基因表达中这一重要步骤的改变是许多人类疾病的基础，因此详细了解所涉及的机制对改善人类健康非常重要。Pre- mRNA剪接由剪接体进行，剪接体是一个由5个小核rna （snrna: U1, U2, U4， U5和U6）和bbb100多肽组成的~ 3mda大分子复合物。虽然在定义这些组成部分方面已经取得了很大的进展，但关于这些无数的部分如何共同作用以精确和及时地介导内含子的去除，还有很多有待了解。本提案描述了将单分子全内反射荧光（SM-TIRF）显微镜应用于剪接体等复杂大分子系统的方法的发展。布兰代斯大学Jeff Gelles的实验室设计并实现了一种具有多波长功能的新型SM-TIRF显微镜，可以同时监测与固定在玻璃盖上的单个荧光前mrna分子相互作用的多个荧光团。使用这种共定位单分子光谱（CoSMoS）方法，可以直接观察剪接体组装和剪接的整个过程中snRNP和剪接因子与前体mrna的关联/解离的详细动力学。由于目前还没有完全重建的系统能够忠实地复制剪接途径的所有阶段，因此所有的实验都是使用荧光标记的内源性蛋白质在粗细胞裂解物中进行的。该方法将用于：(1)在模型pre-mRNA上建立剪接体组装的第一个完整动力学框架；(2)确定剪接体在其他转录本上通过相同或不同的动态途径进行组装的程度；(3)研究剪接体丢弃途径的性质；(4)研究DExH/D-box蛋白与剪接体的动态关系。由于CoSMoS分析不需要纯化组分，可以在高度复杂的混合物中进行，因此该技术应该广泛适用于许多其他大分子机器的研究，如剪接体，不能在体外从组成部分重新组装。

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the detailed molecular mechanisms by which intervening sequences or introns are removed from nascent RNA transcripts through the process of pre-mRNA splicing. Alterations in this essential step in eukaryotic gene expression are known to underly many human diseases, so detailed understanding of the mechanisms involved is important for the betterment of human health. Pre- mRNA splicing is carried out by the spliceosome, a ~3 MDa macromolecular complex consisting of 5 small nuclear RNAs (snRNAs: U1, U2, U4, U5 and U6) and >100 polypeptides. While much progress has been made in defining these component parts, much remains to be learned about how these myriad pieces function together to mediate precise and timely intron removal. This proposal describes development of methodologies for application of single molecule total internal reflection fluorescence (SM-TIRF) microscopy to complex macromolecular systems such as the spliceosome. A novel SM-TIRF microscope with multi-wavelength capabilities designed and implemented in Jeff Gelles' laboratory at Brandeis University allows for simultaneous monitoring of multiple fluorophores interacting with single fluorescent pre-mRNA molecules tethered to a glass coverslip. Using this Colocalization Single Molecule Spectroscopy (CoSMoS) approach, one can directly observe the detailed kinetics of snRNP and splicing factor association/dissociation with pre-mRNA over the full course of spliceosome assembly and splicing. As there currently exists no fully-reconstituted system that faithfully replicates all stages of the splicing pathway, all experiments are carried out in crude cell lysates using fluorescently-tagged endogenous proteins. This approach will be used to: (1) Establish the first complete kinetic framework for spliceosome assembly on a model pre-mRNA; (2) Determine to what extent spliceosome assembly proceeds via the same or different dynamic pathway(s) on other transcripts; (3) Investigate the nature of spliceosomal discard pathways; and (4) Investigate the dynamics of DExH/D-box proteins with the spliceosome. Because CoSMoS analysis does not require purified components and can be carried out in highly complex mixtures, this technology should be broadly applicable to the study of many other macromolecular machines that, like the spliceosome, cannot be reassembled from component parts in vitro. ) PUBLIC HEALTH RELEVANCE: This project seeks to elucidate the molecular mechanisms by which sequences interrupting genes (introns) are removed during the process of gene expression. Such intron splicing is an essential process in all multicellular organisms, and missplicing is a major contributor to many human diseases. Only by gaining a better understanding the cellular machinery mediating this process will we ultimately be able to treat such splicing-related diseases. )