Biosynthesis of RNAs

描述（由申请人提供）：我们的长期兴趣是控制mRNA剪接和输出的特异性和保真度的机制。我们对出芽酵母酿酒酵母的这些问题进行了研究，以开发其巨大的遗传潜力。此外，我们过去20年的工作在验证mRNA剪接的原理方面发挥了重要作用，即基因表达的基本步骤在酵母和哺乳动物之间非常保守。在上一个资助期，我们特别关注了DEAD-box蛋白家族促进的atp驱动重排的作用。我们的研究中出现了一个统一的假设，即mrna通过RNA:RNA或RNA：蛋白质配对伙伴的交换被广泛重塑；在某些情况下，这些配对伙伴是相互排斥的。未来的实验将测试这些依赖ntp的交换不仅在物理上和时间上相关，而且在功能上相互依赖，即耦合的概念。除了明确解决剪接体催化核心的动态设计原则外，这些研究将为基因表达的主要步骤相互整合提供可测试的机制。最后，我们将利用我们最近开发的高通量微阵列技术来评估特定突变和环境胁迫对全套250个内含子基因的全基因组影响。尽管人们普遍认为出芽酵母的剪接不受显著调控，但我们的初步分析表明，这种单细胞生物的剪接实际上受到复杂的组合控制。使用生物化学，遗传和细胞生物学方法的混合，我们提出三个具体目标：1。对拼接第二步催化和保真度的综合模型进行测试确定赋予出口能力的mrnp序列重构中的耦合步骤3。利用全基因组分析鉴定RNA加工中的调控通路

DESCRIPTION (provided by applicant): Our long-term interest is in the mechanisms that control the specificity and fidelity of mRNA splicing and export. We study these problems in the budding yeast Saccharomyces cerevisiae in order to exploit its enormous genetic potential. Moreover, our work over the last 20 years has been instrumental in validating the principle - for mRNA splicing - that the basic steps in gene expression are remarkably conserved from yeast to mammals. In the last funding period, we have focused in particular on the role of ATP-driven rearrangements promoted by the DEAD-box family of proteins. A unifying hypothesis that has emerged from our studies is that mRNPs are remodelled extensively via the exchange of RNA:RNA or RNA:protein pairing partners; in some cases, these pairing partners are mutually exclusive. Future experiments will test the notion that these NTP-dependent exchanges are not only physically and temporally correlated, but are functionally interdependent, i.e. coupled. In addition to explicitly addressing the dynamic design principle of the spliceosome's catalytic core, these studies will suggest testable mechanisms for the integration of the major steps of gene expression with one another. Finally, we will exploit our recent development of highthroughput microarray technology to assess the genome-wide impact of specific mutations and environmental stresses on the full set of 250 intron-containing genes. Despite the widely-held notion that splicing in budding yeast is not significantly regulated, our initial analyses suggest that splicing in this single-cell organism is in fact subject to complex combinatorial control. Using a blend of biochemical, genetic, and cell biological approaches, we propose three Specific Aims: 1. To Test a Comprehensive Model for Catalysis and Fidelity at the Second Step of Splicing 2. To Identify Coupled Steps in the Sequential Remodelling of mRNPs that Confer Export Competence 3. To Identify Regulated Pathways in RNA Processing Using Genome-wide Analysis