BIOSYNTHESIS OF RNAS

我们的长期目标是了解生物学用来控制RNA加工反应的特异性和保真度的策略。在过去的15年里，我们利用酵母遗传学这一强大的工具来分析剪接体的RNA和蛋白质成分。特别是，补偿碱基对分析揭示了催化核心的关键RNA-RNA相互作用，这与其他通过光化学交联获得的数据惊人地一致。综上所述，这些发现使得剪接体是一个惊人的动态大分子机器的结论成为可能。未来的目标集中在我们继续努力了解这些由ATP驱动的结构重组的机制及其调控。此外，我们将探索这样的预测，即剪接的mRNA传递到输出装置，随后从毛孔的细胞质表面释放，也是由eIF4A样ATPase协调的步骤，确保了方向性和选择性。使用分子遗传学、生物化学和细胞生物学的混合方法，我们的实验将解决四个广泛的问题：I.死盒蛋白如何将ATP驱动的RNA重排与保真度联系起来？剪接体催化核心蛋白的作用是什么？如何回收SnRNPs用于新一轮的剪接？四、信使核糖核酸的输出与核糖核酸的加工是如何结合的？我们预测，拟议的实验将对RNP介导的催化、RNA折叠和RNA运输等基本问题提供亟需的见解。在一些情况下，这些发现还有望对与健康有关的问题产生影响，包括逆转录病毒(HIV)RNA新陈代谢和脊髓肌萎缩，这是导致儿童死亡的最常见的遗传原因。

Our long-term goal is to understand the strategies used by biology to control the specificity and fidelity of RNA processing reactions. Over the past 15 years we have exploited the powerful tool of yeast genetics to analyze the RNA and protein components of the spliceosome. In particular, compensatory base pair analysis has revealed critical RNA-RNA interactions at the catalytic core, in striking agreement with data of others obtained by photochemical crosslinking. Taken together, these findings have allowed the conclusion that the spliceosome is a spectacularly dynamic macromolecular machine. Future goals are centered on our continuing efforts to understand the mechanism of these ATP-driven structural rearrangements and their regulation. Additionally, we will explore the prediction that the hand-off of spliced mRNA to the export apparatus, and subsequent release from the cytoplasmic face of the pore, are also steps which are coordinated by eIF4A-like ATPases, ensuring directionality and selectivity. Using a blend of molecular genetic, biochemical and cell biological approaches, our experiments will address four broad questions: I. How do DEAD-box proteins link ATP-driven RNA rearrangements with fidelity? II. What is the role of protein at the spliceosomal catalytic core? III. How are snRNPs recycled for new rounds of splicing? IV. How is mRNA export coupled to RNA processing? We predict that the proposed experiments will shed much-needed insight into fundamental questions about RNP-mediated catalysis, RNA folding and RNA transport. In several cases, these findings also promise to impact on health- related problems, including retroviral (HIV) RNA metabolism, and Spinal Muscular Atrophy, the most common genetic cause of childhood mortality.