Understanding the Functions of a key RNA Base Pair in the Catalytic Core of the Spliceosome

摘要 前体mRNA剪接是人类基因表达的一个重要步骤， RNA-蛋白质复合物称为剪接体。了解的结构和功能， 剪接体对于剪接相关疾病如视网膜炎的治疗方法的发展至关重要 色素沉着症和骨髓增生异常综合征，并将提供深入了解RNA和蛋白质如何合作， 执行细胞功能。U6 RNA是剪接体的核心成分， 在每个剪接事件期间构象和结合配偶体的重排。我们的目标是确定 U6 RNA的结构赋予其在整个剪接周期中的功能，并将使用遗传上易于处理的 芽殖酵母酿酒酵母作为我们的模型系统。基于生物化学和遗传学证据， 我们假设高度保守的U6核苷酸A62和C85参与重要的RNA-RNA 和/或RNA-蛋白质相互作用的剪接周期，尚未得到表征。确定 这些残留物的功能，我们将追求三个具体目标。在目标1中，我们将使用生物化学测定， 确定突变U6-A62U/C85A（U6-UA）和U6-A62C/C85G（U6-CG）的阻滞点，并将 进行一项遗传实验，将突变体的缺陷与剪接体解旋酶的功能联系起来。在aim中 2，我们将使用全基因组选择寻找抑制U6-UA和U6-CG缺陷的突变， 选择靶向剪接体的"主调节器"蛋白Prp8。在目标3中，我们将使用诱变， 体内光交联和下拉测定以确定Prp8突变如何抑制U6-UA缺陷。 这项实验将由已发表的模型中的丰富结构信息指导。 通过冷冻电子显微镜解决剪接体复合物。我们提出的研究有可能（i） 确定一个新的冷敏感块在剪接周期，提供了一个实验工具，以更好地 了解剪接的机制，以及（ii）揭示了重要的分子相互作用级联，以确保 精确拼接。

Abstract Pre-mRNA splicing, an essential step in human gene expression, is catalyzed by a large and dynamic RNA-protein complex called the spliceosome. An understanding of the structure and function of the spliceosome is critical to the development of therapeutics for splicing-associated diseases such as retinitis pigmentosa and myelodysplastic syndromes, and will provide insight into how RNAs and proteins cooperate to carry out cellular functions. U6 RNA is a core component of the spliceosome and undergoes dramatic rearrangements in conformation and binding partners during each splicing event. We aim to determine how the structure of U6 RNA confers its function throughout the splicing cycle, and will use the genetically tractable budding yeast Saccharomyces cerevisiae as our model system. Based on biochemical and genetic evidence, we hypothesize that the highly conserved U6 nucleotides A62 and C85 participate in important RNA-RNA and/or RNA-protein interactions in the splicing cycle that have not yet been characterized. To determine the function(s) of these residues, we will pursue three specific aims. In Aim 1, we will use biochemical assays to determine the arrest point of the mutations U6-A62U/C85A (U6-UA) and U6-A62C/C85G (U6-CG), and will conduct a genetic experiment to relate the mutants’ defects to the function of a spliceosomal helicase. In Aim 2, we will look for mutations that suppress the defects of U6-UA and U6-CG using genome-wide selections and a selection targeted to the spliceosome’s “master regulator” protein Prp8. In Aim 3, we will use mutagenesis, in vivo photocrosslinking, and pull-down assays to determine how mutations in Prp8 suppress the U6-UA defect. This experimentation will be guided by the wealth of structural information available in published models of spliceosomal complexes solved by cryo-electron microscopy. Our proposed research has the potential to (i) identify a novel cold-sensitive block in the splicing cycle, providing an experimental tool with which to better understand the mechanism of splicing, and (ii) reveal a cascade of molecular interactions important to ensure accurate splicing.