Structural and Functional Studies of Spliceosome Assembly and Activation

真核生物中的前信使RNA(pre-mRNAs)是一种分裂基因结构，编码外显子序列由非编码内含子序列分隔。内含子被切除和外显子连接在一起的过程被称为前mRNA剪接，由RNA-蛋白质复合体-剪接体催化，剪接体从酵母到人类都高度保守。虽然组成性剪接事件的忠实执行是功能mRNAs成熟所必需的，但选择性剪接事件在高等真核细胞的分化和发育以及病毒的生命周期中代表了基因调控的基本水平。 剪接体由U1、U2和U4/U5/U6核糖核蛋白小颗粒组成，每个小核核糖核蛋白颗粒都含有一个独特的RNA和相关蛋白，它们以有序的方式组装在前mRNA底物上。虽然整个组装途径是已知的，但调控剪接体组装和激活的分子机制仍然知之甚少。我们正在利用各种生化、化学和生物物理技术来研究剪接体组装的各个步骤以及成熟剪接体中的转变。 我们目前的研究重点是基于剪接体SF3b14(P14)和Prp8的两个关键蛋白质组分的鉴定，这两个组分都识别前mRNA中的关键反应序列，并且对剪接体组装和调节剪接体内的催化活性至关重要。这些蛋白质直接接触前mRNA底物和剪接体RNA，招募或形成人类剪接体的核心。我们已经解决了p14和Prp8的一个关键结构域(RNaseH或RH结构域)的高分辨率X射线结构，并利用生物化学、酵母遗传学和细胞生物学对这两个因素进行了研究。我们的结构反过来又提出了这些蛋白质的功能模型，可以通过进一步的实验进行测试。我们提出的研究包括四组实验：(1)p14RNA相互作用的生化/结构研究(2)细胞和细胞提取物中p14在剪接中的作用；(3)评估Prp8-RH结构域中的局部构象开关，揭示关键的金属结合位点；(4)通过基于结构的突变研究Prp8的大规模构象变化。 这些研究将为重要的基因调控途径的基本方面提供新的见解，包括剪接体组装的机制和剪接体在剪接的化学步骤中重排的性质和要求。它们还将扩大我们对蛋白质和RNA结构以及细胞内RNA蛋白质相互作用的知识，这些知识在从转录到RNA加工本身再到翻译的各种过程中都是至关重要的。

Pre-messenger RNAs (pre-mRNAs) in eukaryotes are characterized by a split-gene structure where coding exon sequences are separated by non-coding intron sequences. The process by which introns are excised and exons are joined together is known as pre-mRNA splicing and is catalyzed by an RNA-protein complex, the spliceosome, that is highly conserved from yeast to humans. While the faithful execution of constitutive splicing events is required for the maturation of functional mRNAs, alternative splicing events represent a fundamental level of gene regulation in terms of differentiation and development in higher eukaryotic cells and in the life cycle of viruses. The spliceosome consists of the U1, U2, and U4/U5/U6 snRNPs (small nuclear ribonucleoprotein particles) each containing a unique RNA and associated proteins which assemble on pre-mRNA substrates in an ordered fashion. Although the overall assembly pathway is known, the molecular mechanisms which govern spliceosome assembly and activation remain poorly understood. We are utilizing a variety of biochemical, chemical, and biophysical techniques to study the individual steps of spliceosome assembly as well as transitions in the mature spliceosome. The current focus of our studies is based on the characterization of two critical protein components of the spliceosome SF3b14 (p14) and PRP8, both of which recognize key reactive sequences within the pre-mRNA and are critical for spliceosome assembly and regulating the catalytic activity within the spliceosome. These proteins directly contact the pre-mRNA substrate and spliceosomal RNAs recruiting or forming the core of the human spliceosome. We have solved the high-resolution X-ray structures of both p14 and a key domain of PRP8 (the RNase H or RH domain) and investigated both of these factors using biochemistry, yeast genetics, and cell biology. Our structures in turn suggest models for the function of these proteins that can be tested by further experimentation. Our proposed studies involve four sets of experiments: (1) biochemical/structural investigation of p14RNA interactions (2) characterization in cells and cellular extract of the role of p14 in splicing; (3) assessment of a local conformational switch in the PRP8-RH domain that unmasks a critical metal-binding site; (4) investigation of large-scale conformational change in PRP8 by structure-based mutagenesis. These studies will provide novel insights into fundamental aspects of an essential gene regulatory pathway pre-mRNA splicing including mechanisms of spliceosome assembly and the nature and requirements of spliceosomal rearrangements during the chemical steps of splicing. They will also expand our knowledge of protein and RNA structure and RNAprotein interaction within the cell which are critical in diverse processes ranging from transcription through RNA processing itself to translation.