Grant: Discovery Grants Program - Individual

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.