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前信使核糖核酸剪接过程从新生RNA转录本中移除插入序列或内含子的详细分子机制。真核基因表达的这一重要步骤的改变是已知的许多人类疾病的基础，因此详细了解其中涉及的机制对于改善人类健康非常重要。剪接体是由U1、U2、U4、U5和U6 5个小核RNA和&gt；100多肽组成的~3MDA大分子复合体，通过剪接体进行mRNA的剪接。虽然在确定这些组成部分方面已经取得了很大进展，但关于这些无数片段如何共同发挥作用以调节准确和及时的内含子去除，仍有许多有待了解。这项提案将应用两种新的使能方法-RIPiT-Seq(RNAIP串联结合深度测序)和COSMOS(共定位单分子光谱)来研究哺乳动物剪接体的体内和体外组装。RIPiT-Seq允许研究人员在体内绘制整个转录组中具有特定组成的多组分RNPs的占据位置。通过改变用于IP和/或亲和纯化+/-甲醛交联的蛋白质对，可以确定不同络合物的完整占据情况，以及哪些结合位点是动力学稳定的，哪些是更动态的。在光谱的另一端，COSMOS可以实时观察单个前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：保留的和缓慢处理的内含子上的剪接体组装状态是什么？选择性剪接决定是否仅限于早期复合体，或者某些决定是否涉及后期复合体？除了目前定义的内含子末端之外，是否还有哺乳动物剪接体催化的位点？哺乳动物剪接体组装和酵母剪接体组装一样有活力吗？在大规模转录重编程过程中，剪接体组装途径如何改变以有利于包含或排除盒式磁带外显子？总之，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的理解，以及这些途径如何改变以调节基因表达输出对外部刺激的反应。

 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.