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

Northwestern University, Evanston IL

Investigators

Abstract

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.