The molecular mechanism of U6 snRNA activation for pre-mRNA splicing

生物所做的最基本的活动之一是将它们的遗传信息（DNA）复制到蛋白质中，蛋白质是执行生物体生存所需的许多功能的分子机器。DNA复制成蛋白质分两步进行：首先，DNA被复制成RNA（类似于DNA），其次，RNA被用作指定特定蛋白质的模板。DNA包含一段“非编码”信息——不包含制造蛋白质信息的区域——必须在蛋白质合成前移除。如果不能去除非编码区，就会导致蛋白质受损，无法发挥作用，事实上，这往往与癌症等疾病有关。非编码序列在RNA阶段被移除，这一过程被称为前mrna剪接。这项提议的目的是了解执行剪接反应的机制如何将自己组装到目标RNA上，被激活，并导致剪接事件发生。这个过程中的一个重要步骤是被称为U6的中心成分被另一个称为U4的粒子激活。尽管在剪接方面进行了三十年的研究，但U4颗粒从未被生物化学纯化到足够大的数量来表征。这里提出的实验利用了Rader实验室开发的一种新的U4纯化程序来研究颗粒的组成（即与其相关的蛋白质），其形状以及它如何识别和结合U6。利用强大的生物物理工具，这些实验将揭示U4和U6在相互作用时如何改变形状，以及形状变化如何使U6组装到拼接机制中。作为仅发生在两个粒子上的剪接相关组装步骤之一，U4和U6的相互作用为理解蛋白质和rna在这些复杂和动态粒子中的相互作用和功能提供了一个方便的模型。

One of the most basic activities that living things do is to copy their genetic information (DNA) into proteins, the molecular machines that carry out many of the functions that an organism requires to live. The copying of DNA into proteins takes place in two steps: first, DNA is copied into RNA (which is similar to DNA), and, second, RNA is used as a template to specify a particular protein. The DNA includes stretches of "non-coding" information - regions that do not contain information for making proteins - that must be removed before protein synthesis. Failure to remove the non-coding regions leads to corrupted proteins that do not work and, in fact, are frequently associated with diseases such as cancer. Non-coding sequences are removed during the RNA stage in a process known as pre-mRNA splicing. The object of this proposal is to learn how the machinery that carries out the splicing reaction assembles itself onto a target RNA, becomes activated, and causes the splicing events to occur. An important step in this process is the activation of a central component, known as U6, by another particle called U4. Despite three decades of work on splicing, the U4 particle has never been purified biochemically in large enough quantities to characterize. The experiments proposed here take advantage of a novel U4 purification procedure developed by the Rader lab to investigate the composition of the particle (i.e. which proteins are associated with it), its shape, and how it recognizes and associates with U6. Using powerful biophysical tools, these experiments will reveal how U4 and U6 change shape as they interact, and how the shape changes allow U6 to assemble into the splicing machinery. As one of the few splicing-related assembly steps that occurs with just two particles, the interaction of U4 and U6 provides a convenient model for understanding how proteins and RNAs interact and function in these complex and dynamic particles.