Pre-mRNA Splicing Mechanisms

描述（由申请人提供）：本项目的长期目标是阐明通过前体mRNA剪接过程从新生RNA转录物中去除插入序列或内含子的详细分子机制。已知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至关重要。前mRNA剪接是由剪接体进行的，剪接体是由5个小核RNA（snRNA：U1、U2、U4、U 5和U6）和>100个多肽组成的~3 MDa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进展，但关于这些无数的片段如何共同发挥作用以调节精确和及时的内含子去除，还有很多东西需要了解。该提案描述了单分子全内反射荧光（SM-TIRF）显微镜应用于复杂的大分子系统，如剪接体的方法的发展。在布兰迪斯大学的Jeff Gelles实验室中设计和实施的具有多波长功能的新型SM-TIRF显微镜允许同时监测与固定在玻璃盖玻片上的单个荧光前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snRNP和剪接因子在剪接体组装和剪接的整个过程中与前体mRNA缔合/解离的详细动力学。由于目前不存在忠实地复制剪接途径的所有阶段的完全重构系统，所有实验都使用荧光标记的内源性蛋白质在粗细胞裂解物中进行。这种方法将用于：（1）建立第一个完整的动力学框架，用于在模型前mRNA上进行剪接体组装;（2）确定剪接体组装在其他转录物上通过相同或不同的动态途径进行的程度;（3）研究剪接体丢弃途径的性质;以及（4）研究DExH/D-box蛋白与剪接体的动力学。由于CoSMoS分析不需要纯化的组分，并且可以在高度复杂的混合物中进行，因此该技术应广泛适用于许多其他大分子机器的研究，这些机器如剪接体，不能在体外从组成部分重新组装。) 公共卫生相关性：该项目旨在阐明基因表达过程中中断基因（内含子）序列被移除的分子机制。这种内含子剪接是所有多细胞生物体中的一个重要过程，而错误剪接是许多人类疾病的主要原因。只有更好地了解介导这一过程的细胞机制，我们最终才能治疗此类剪接相关疾病。)

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. )