Structural and Functional Studies of Spliceosome Assembly and Activation
剪接体组装和激活的结构和功能研究
基本信息
- 批准号:RGPIN-2016-05175
- 负责人:
- 金额:$ 2.77万
- 依托单位:
- 依托单位国家:加拿大
- 项目类别:Discovery Grants Program - Individual
- 财政年份:2019
- 资助国家:加拿大
- 起止时间:2019-01-01 至 2020-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
真核生物的前信使rna (pre- mrna)具有基因分裂结构,编码外显子序列被非编码内含子序列分开。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,它是由rna -蛋白质复合物剪接体催化的,这种剪接体从酵母到人类都是高度保守的。虽然组成剪接事件的忠实执行是功能性mrna成熟所必需的,但可选剪接事件代表了高级真核细胞和病毒生命周期中分化和发育的基因调控的基本水平。******剪接体由U1, U2和U4/U5/U6 snRNPs(小核核糖核蛋白颗粒)组成,每个snRNPs含有一个独特的RNA和相关的蛋白质,它们以有序的方式组装在mrna前底物上。虽然整个组装途径是已知的,但控制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物化学、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的转变。******我们目前的研究重点是基于剪接体的两个关键蛋白组分SF3b14 (p14)和PRP8的表征,这两个蛋白组分都识别pre-mRNA中的关键反应序列,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(RNase H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研究了这两个因素。我们的结构反过来为这些蛋白质的功能提供了模型,可以通过进一步的实验来测试。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在剪接中的作用的表征;(3)评估PRP8-RH结构域的局部构象开关,该开关揭示了一个关键的金属结合位点;(4)基于结构诱变的PRP8大规模构象变化研究。******这些研究将为基本基因调控途径pre-mRNA剪接的基本方面提供新的见解,包括剪接体组装的机制以及剪接化学步骤中剪接体重排的性质和要求。他们还将扩大我们对蛋白质和RNA结构以及细胞内RNA -蛋白质相互作用的认识,这在从转录到RNA加工本身到翻译的各种过程中都是至关重要的。
项目成果
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Macmillan, Andrew的其他文献
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{{ truncateString('Macmillan, Andrew', 18)}}的其他基金
Structural and Functional Studies of Spliceosome Assembly and Activation
剪接体组装和激活的结构和功能研究
- 批准号:
RGPIN-2016-05175 - 财政年份:2021
- 资助金额:
$ 2.77万 - 项目类别:
Discovery Grants Program - Individual
Structural and Functional Studies of Spliceosome Assembly and Activation
剪接体组装和激活的结构和功能研究
- 批准号:
RGPIN-2016-05175 - 财政年份:2020
- 资助金额:
$ 2.77万 - 项目类别:
Discovery Grants Program - Individual
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