CAREER: A New Approach For Analysis of RNA Dynamics During Pre-mRNA Splicing

0093003埃里克·桑特海默二十多年来，人们一直知道，功能性信使核糖核酸不是DNA的原始拷贝，而是一种高度编辑的分子，其中非功能部分(内含子)被精确地“拼接”掉。基因信息的忠实表达要求内含子的去除具有显著的准确性。剪接机制由五个RNA分子(以及几十个蛋白质)组成一个极其动态的复合体，在这个复合体中，多个RNA相互作用形成、断裂和重排。尽管进行了密集的努力，但这种结构可塑性背后的机械原理仍然模糊不清，很大程度上是因为无法直接和具体地控制个人的重新安排。在这个职业项目的研究部分，研究人员将开发一种新的生化技术，允许在复杂的含蛋白质环境中对RNA结构进行可逆的共价固定。这种方法利用了砷和硫原子之间的高亲和力。将两个硫原子加入到RNA双链的每条链中，将为含有两个砷原子的化合物产生一个四硫结合位点(“双砷”)。然后，研究人员将开发双砷化合物，这种化合物可以通过添加过量的含有两个硫原子的解毒剂来逆转修饰的RNA。一旦交联系统被开发和表征，它将被用来捕获剪接机器中的动态RNA相互作用。这将允许对在剪接途径的离散阶段停滞不前的剪接复合体进行生化表征。交联链的可逆性将使研究人员能够确定被认为控制剪接体中RNA重排的酶的作用，包括先前涉及促进准确剪接位点选择的蛋白质。在这个项目过程中开发的技术将适用于许多其他涉及核酸重排的系统。在过去的二十年里，人们也清楚地看到，细胞的许多中心工作涉及大分子组装，这些组装使许多酶相形见绌，这些酶在结构和机械上都有很好的特征。为了理解类似深度的基因表达，必须面对这种规模和复杂性带来的挑战。生物二年级和三年级学生所需的大部分生物化学课程提供了相对简单和定义明确的生化系统的坚实基础，主要来自中间代谢。然而，真核基因表达的过程往往是以更多的描述性、较少的量化和机械性的方式传授的。随着基因表达领域的发展超越了“部分清单”阶段，本科生物化学课程应该反映这一点，纳入最令人兴奋的当前研究，并激发人们对解决仍然存在的一些重大挑战的兴趣。在这个职业奖项的教育部分，研究人员将开发一门初级生物化学课程，像以前一样，涵盖大分子结构、功能和能量学的基础，但它的许多插图和例子来自分子和细胞生物学领域，这些领域是当前紧张调查的主题。这种方法的预期目标是强调当前该领域的兴奋，使学生为后基因组时代等待生物化学家的爆炸性机会做好准备，并向他们灌输解决更大和更复杂系统的工作所必需的创造力和跨学科。

0093003 Erik Sontheimer It has been known for over two decades that functional messenger RNA is not a raw copy of DNA but is instead a highly edited molecule wherein non-functional portions (introns) are precisely "spliced" away. The faithful expression of genetic information requires that intron removal occur with remarkable accuracy. The splicing machinery consists of five RNA molecules that (along with several dozen proteins) comprise an extremely dynamic complex in which multiple RNA interactions form, break, and rearrange. Despite intensive efforts, the mechanistic principles that underlie this structural plasticity have remained obscure, largely because of the inability to control individual rearrangements directly and specifically. In the research component of this CAREER project, the investigator will develop a new biochemical technique that allows the reversible covalent fixation of RNA structure within a complex protein-containing environment. This approach takes advantage of the high affinity between arsenic and sulfur atoms. Incorporation of two sulfur atoms into each strand of an RNA duplex will generate a four-sulfur binding site for a compound containing two arsenic atoms ("biarsenical"). The investigator will then develop biarsenical compounds that can crosslink the modified RNAs in a way that can be reversed upon addition of an excess of antidote containing two sulfur atoms. Once the crosslinking system has been developed and characterized, it will be used to trap dynamic RNA interactions within the splicing machinery. This will then permit the biochemical characterization of splicing complexes that are stalled at discrete stages of the splicing pathway. The reversibility of the crosslinks will allow the investigator to define the roles of enzymes thought to control RNA rearrangements in the spliceosome, including proteins previously implicated in promoting accurate splice site selection. The techniques developed in the course of this project will be applicable to many other systems that involve nucleic acid rearrangements. Over the past twenty years, it has also become clear that many central workings of the cell involve large macromolecular assemblies that dwarf many of the enzymes that have been so well characterized structurally and mechanistically. To understand gene expression at a similar depth, the challenges presented by this size and complexity must be faced. Much of the biochemistry curriculum required of sophomore- and junior-level biology students provides a solid grounding in relatively simple and well-defined biochemical systems, predominantly from intermediary metabolism. The processes of eukaryotic gene expression, however, have often been taught in a more descriptive and less quantitative and mechanistic fashion. As the field of gene expression progresses beyond the "parts list" phase, undergraduate biochemistry courses should reflect this, to incorporate the most exciting current research and to spur interest in addressing some of the great challenges that remain. In the educational component of this CAREER award, the investigator will develop a junior-level biochemistry course that (as before) covers the fundamentals of macromolecular structure, function, and energetics, but that draws many of its illustrations and examples from areas of molecular and cell biology that are the subjects of intense current investigation. The intended goal of this approach is to emphasize the current excitement in the field, to prepare students for the exploding opportunities that await biochemists in the post-genome era, and to impress upon them the creativity and interdisciplinarity that will be necessary to tackle the workings of larger and more complex systems.