Determining the role of lysine acetylation in alternative splicing regulation - The process of alternative splicing is regulated in part by RNA binding proteins (RBPs) that bind to corresponding sequence elements in the gene transcript and influence the assembly of the spliceosome components at adjacent splice sites. Recent studies highlight that many RBPs are modified at lysine side chains by adding acetyl groups. Acetylation neutralizes the positive charge of the lysine side chain, which can i) disrupt its capacity to interact with the negatively charged RNA backbone, ii) disrupt interactions with the π electron cloud in nitrogenous bases, and iii) alter its ability to interact with other proteins. Acetylated RBPs have been identified in many cancers and are linked to proteinopathy in neurodegenerative diseases. Similarly, spliced variants have been identified in many gene transcripts related to neurodegenerative diseases and cancers. Collectively, these data suggest a role for RBP acetylation in generating these disease-related spliced variants. The treatment of such splicing-related diseases would be facilitated by a detailed mechanistic understanding of how lysine acetylation regulates RBP properties and, in turn, alternative pre-mRNA splicing to generate spliced variants. In this project, we propose to investigate the role of acetylation in alternative splicing regulation using the RBP polypyrimidine tract binding protein 1 (PTBP1). PTBP1 is a well-characterized, multi-valent RBP involved in alternative splicing regulation. PTBP1 is acetylated at several lysine residues in each of the four RBDs and the N-terminal region that contains overlapping nuclear localization and export sequences. PTBP1 most often functions to repress the inclusion of regulated exons in spliced mRNA, and the mechanism of PTBP1- mediated splicing regulation is well understood. We determined that PTBP1 acetylated lysine residues are highly conserved and participate in RNA binding, protein-protein interactions, and sub-cellular localization. Thus, we hypothesize that acetylation regulates the splicing activity of PTBP1. Our specific aims are to test this hypothesis. Aim1: Determine the role of PTBP1 RBD acetylation in its splicing activity Aim2: Examine the role of acetylation in PTBP1 cellular localization and, in turn, splicing activity. Aim3: Investigate signaling networks and transcriptome-wide targets of acetylated PTBP1 Our studies will answer fundamentally important questions about how acetylation modulates alternative splicing activity to dictate mRNA isoforms, including disease-related splice variants. Importantly, our studies can potentially unravel a novel mode of gene regulation at the level of mRNA splicing in eukaryotes. The proposed studies will leverage experimental approaches established in the laboratory by previous undergraduate trainees and provide new students (including those from backgrounds underrepresented in biomedical sciences) an excellent opportunity to gain hands-on research experience in splicing chemical biology.
