Julia R. Widom, Ph.D. - US grants

? DESCRIPTION (provided by applicant): A surprising discovery was made in the 1970s when researchers found that in many higher organisms, such as humans, only a small fraction of the genome encodes proteins, whereas the rest is seemingly junk DNA. In addition, the vast majority of genes have their protein-coding regions (exons) split up, separated by introns containing up to tens of thousands of nucleotides. We now know that non-protein-coding regions of the genome encode RNAs that contribute to gene regulation, catalysis and more, and the introns separating coding regions of genes are removed and the exons are joined together in a process called splicing. This critical step in gene expression allows for exquisitely fine-tund regulation and, through alternative splicing, allows a single gene to encode for more than one protein. Mistakes in this process can be lethal - it has been estimated that up to 60% of human genetic diseases involve defects in splicing. Splicing is executed by the spliceosome, a multi-megaDalton macromolecular machine whose function depends on the interplay between many protein and RNA components. Determining the roles of and interactions between these components is of central importance to understanding the process of splicing and, therefore, the molecular mechanisms of the many human diseases in which splicing is implicated. This proposal focuses on two proteins, Prp22 and Prp16, which facilitate structural rearrangements in the yeast spliceosome (which is very similar to that found in humans). These proteins function as RNA helicases in vitro, but it is not known how this helicase activity contributes to their function in the spliceosome. Specific Aim 1 involves studying the changes to pre-mRNA conformation induced by Prp22, separately examining its roles in the second catalytic step of splicing and in mRNA product release after the second step. Specific Aim 2 focuses on Prp16, which has been shown to play a proofreading role, triggering the discard of suboptimal pre- mRNA substrates prior to step 1 of catalysis. The approach will utilize the tools of single-molecule fluorescence resonance energy transfer (smFRET), which will provide the sensitivity to compare not only the pre-mRNA conformations present in different intermediate states in splicing, but also their dynamics. By introducing blocks in the splicing cycle using dominant negative mutations in Prp22 and Prp16, the changes in pre-mRNA conformation and dynamics induced by these proteins will be measured. An important facet of this work will be comparing the helicase/ATPase activities of Prp16 and Prp22 on model substrates in solution to their activities in the spliceosome. The knowledge provided by this project will be relevant to the understanding and treatment of diseases that involve defects in splicing. In addition, the project will provide important training in the areas of RNA biophysics, single-molecule spectroscopy, and scientific writing, presentation and mentoring.

Project Summary In eukaryotes, the vast majority of genes have their protein-coding regions (exons) split up, separated by introns containing up to tens of thousands of nucleotides. The removal of introns, called ?splicing?, is a critical step in gene expression that allows for exquisitely fine-tuned regulation and, through alternative splicing, diversifies a single gene into more than one protein. Splicing is executed by the spliceosome, a multi- megaDalton macromolecular complex whose function requires interactions between the pre-messenger RNA (pre-mRNA) substrate, five small nuclear ribonucleoprotein particles (snRNPs), and numerous additional protein factors. Determining the roles of and interactions between these components is of central importance to understanding the molecular mechanisms of the many human diseases in which aberrant splicing is implicated. The recent application of single-molecule microscopy to the spliceosome has shed much light on the molecular mechanism of splicing. However, the interactions between the snRNAs and the pre-mRNA have remained difficult to probe due to the challenge of preparing snRNAs that are site-specifically fluorophore- labeled. Furthermore, conformational changes can be tracked only on certain length scales, limited by the sensitivity of the experimental techniques used, which are often based on Förster resonance energy transfer (FRET). To address these challenges, Specific Aim 1 will study the rearrangement of interactions between U5 snRNA and the pre-mRNA in response to the action of RNA helicase Prp22. Site-specifically fluorophore- labeled U5 will be prepared through by using a short peptide nucleic acid oligomer to stall transcription by T7 RNA polymerase at the desired labeling site, a general approach that avoids many of the downsides of other RNA labeling methods. Specific Aim 2 proposes the novel technique of FRET-filtered spectroscopy (FFS), which will utilize two closely spaced fluorophores as a FRET donor, and an additional fluorophore as an acceptor. FFS will use electronic coupling between the two donors to reveal their local conformation as a function of their distance from the acceptor, and can be expanded to utilize any type of fluorescence-detected spectroscopy as a readout. This technique will be applied to Cy3- and Cy5-labeled RNA to study the unwinding of RNA duplexes by Prp22. Specific Aim 3 combines the labeling method of Aim 1 with FFS, utilizing FRET- filtered circular dichroism spectroscopy to determine the changes in local pre-mRNA conformation in the vicinity of the branchpoint adenosine as purified Bact intermediates are chased through the first step of splicing. This work will answer longstanding questions about the correlations between local and global RNA conformations in the spliceosome, and involves novel methods that can be generalized to many different biological systems. Aim 1 and the initial experiments for Aim 2 will be pursued in the laboratory of the applicant's research mentor, while Aim 2 will be completed and Aim 3 will be both initiated and completed in the applicant's independent laboratory. During the mentored phase of the award, the applicant will be working at the University of Michigan in the laboratory of Dr. Nils Walter, who has a strong record of training successful scientists. The applicant has assembled an advisory committee who, together with Dr. Walter, will provide guidance on her research and her transition into an independent career. The applicant's career goals involve running an independent laboratory at an academic institution, and she seeks to combine her graduate training in spectroscopy with her ongoing postdoctoral training in RNA molecular biology and biophysics. In addition to providing the instrumentation necessary for the proposed research, the University of Michigan hosts numerous organizations and events that will contribute to the applicant's training and career development. This proposal builds on all of the applicant's previous and ongoing training to open a unique window into the function of the spliceosome.