Biological function and regulation of PRMT9 in synapse development

Project Summary The goal of this project is to determine the molecular mechanisms by which pre-mRNA splicing is regulated by protein arginine methylation, one of the most abundant post-translational modifications on the RNA binding proteins, and to define the impact of this regulation in synapse development and brain function. The human genome encodes nine protein arginine methyltransferases (PRMT1–9), which catalyze three types of arginine methylation: monomethylation (MMA), asymmetric dimethylation (ADMA), and symmetric dimethylation (SDMA). A few years ago, we characterized the newest member of the PRMT family–PRMT9 and identified the splicing factor SF3B2 as the primary substrate of PRMT9, linking its function to pre-mRNA splicing. However, the biological function of PRMT9 and the molecular mechanism by which PRMT9-catalyzed SF3B2 arginine methylation regulates pre-mRNA splicing are still unknown. Recently, mutation of PRMT9 was identified in autosomal recessive intellectual disability (ID) from a large whole genome sequencing study of 136 consanguineous families, underscoring the significance of understanding the biological function and regulation of this newest PRMT. In our preliminary studies, we determined that the ID-associated PRMT9 mutation abolishes its arginine methyltransferase activity on SF3B2, and the mutant protein is unstable and subject to heavy ubiquitination. By establishing a novel Prmt9 conditional knockout mouse model, we revealed that PRMT9 loss causes abnormal synapse development and impairs mouse learning and memory. Mechanistically, we discovered a critical protein-RNA interaction between the arginine 508 (R508) of SF3B2, the site that is exclusively methylated by PRMT9, and the pre-mRNA anchoring site, a cis-regulatory element located upstream of the branch point sequence (BPS). Additionally, we uncovered two molecular pathways that regulate PRMT9 expression at the mRNA and protein levels. Built upon these exciting discoveries, our central hypothesis is that PRMT9-mediated arginine methylation regulates SF3B2–anchoring site interaction and U2 snRNP recruitment, a process that is critical for pre-mRNA splicing and brain development. This hypothesis will be tested in three specific aims: 1) determine how PRMT9-mediated SF3B2 arginine methylation regulates pre-mRNA splicing; 2) define the molecular pathways that regulate PRMT9 expression; and 3) investigate how PRMT9 loss-of-function impacts synapse development and circuit connectivity. Successful completion of these proposed studies will reveal a novel and fundamental molecular network underlying the regulation of RNA splicing in brain development. The key pathways revealed in this study could be harnessed as targets for treating neurodevelopmental disorders.