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。该提案将应用两种新的使能方法，RIPiT-Seq（RNA IP串联与深度测序相结合）和CoSMoS（共定位单分子光谱），以研究哺乳动物体内和体外剪接体组装。RIPiT-Seq允许研究人员在整个转录组中绘制具有确定组成的多组分RNP的体内占用位点。通过改变用于IP和/或亲和纯化+/-甲醛交联的蛋白质对，可以确定不同复合物的完整占用景观，以及哪些结合位点是动力学稳定的，哪些是更具动态性的。在光谱的另一端，CoSMoS能够真实的时间观察单个前mRNA分子上的剪接体组装动力学。拟议的实验将解决许多关键问题，包括：什么是剪接体组装状态保留和缓慢加工的内含子？选择性拼接决策是否严格限于早期阶段的复合体，或者某些决策是否涉及后期阶段的复合体？除了目前定义的内含子末端，哺乳动物剪接体催化的位点是否存在？哺乳动物剪接体的组装是否像酵母剪接体组装一样动态？剪接体组装途径以何种方式改变，以利于在大规模转录重编程时包含或排除盒式外显子？这些实验将从根本上改变我们对哺乳动物剪接体组装的基本途径以及这些途径如何改变以调节基因表达输出以响应外部刺激的理解。

 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 will apply two new enabling methodologies, RIPiT-Seq (RNA IP in Tandem combined with Deep Sequencing) and CoSMoS (Colocalization Single Molecule Spectroscopy), to study mammalian spliceosome assembly in vivo and in vitro. RIPiT-Seq allows researchers to map in vivo occupancy sites of multicomponent RNPs of defined composition across the entire transcriptome. By varying the pairs of proteins used for IP and/or affinity purification +/- formaldehyde crosslinking one can determine the complete occupancy landscapes for different complexes, as well as which binding sites are kinetically stable and which are more dynamic. At the other end of the spectrum, CoSMoS enables real time observation of spliceosome assembly dynamics on single pre-mRNA molecule. The proposed experiments will address numerous key questions including: What is the spliceosome assembly state on retained and slowly processed introns? Are alternative splicing decisions strictly limited to early stage complexes, or do some decisions involve later stage complexes? Are there any sites of mammalian spliceosome catalysis at sites other than currently defined intron ends? Is mammalian spliceosome assembly as dynamic as yeast spliceosome assembly? In what way are spliceosome assembly pathways altered to favor inclusion or exclusion of cassette exons upon large-scale transcriptional reprogramming? Together these experiments will fundamentally change our understanding of the basic pathways of mammalian spliceosome assembly and how these pathways are altered to tune gene expression output in response to external stimuli.