The Characterization of RNA Binding Motif Protein 48 (RBM48) in U12 Intron Splicing, Cellular Differentiation, and Development

About This Grant

PROJECT SUMMARY The project aims to improve our understanding of the role of a novel RNA Binding Motif Protein 48 (RBM48) and its impact on cell proliferation and differentiation. RBM48 represents a core minor spliceosomal protein essential for the splicing of minor or U12-type introns, constituting a small fraction of deeply conserved introns. The splicing of U12 introns has a similar role in cell differentiation across divergent eukaryotes. However, the mechanism by which U12 splicing controls these cell differentiation pathways is poorly understood. Our prior work demonstrated that the disruption of orthologous RBM48 in humans and plants disrupts the splicing of multiple minor intron-containing genes (MIGs). The aberrant splicing of U12 introns in human hematopoietic stem and progenitor cells (HSPCs) adversely impacts normal myeloid cell differentiation, which is proposed to be associated with myelodysplastic syndrome and subsequent onset of acute myeloid leukemia. Human RBM48 encodes multiple transcript isoforms by alternative splicing; however, the biological relevance of this process and the role of transcript isoforms is unclear. The proposal aims to implement cellular, genetic, genomic, and bioinformatic tools to investigate the mechanistic role of RBM48 and its transcript isoforms in U12 splicing. The proposal will also examine how RBM48 modulates U12 splicing in HSPCs, directly affecting differentiation, engraftment potential, and self-renewal of this stem cell population. The data generated will no doubt bridge the gap in our understanding of a growing number of diseases ranging from hematological and neurological to cancer that have been linked to defects in U12 splicing. In Specific Aim 1, we will use a developed genetic assay in human K562 leukemia cells to determine the functional role of RBM48 transcript isoforms in U12 splicing and implement mRNA-seq-based transcriptomic data to determine impacted genes and downstream processes that regulate U12 splicing mediated cellular differentiation. In vivo and in vitro protein-protein interactions of RBM48 transcript isoforms will be used to elucidate the mechanistic role in U12 splicing. The data will lead to a better definition of U12 intron consensus and potentially identify sequence targets of RBM48. We have shown that RBM48 knockdown alters the survival and proliferation characteristics of K562 cells. In Specific Aim 2, we will use an RBM48 knockdown strategy in HSPCs to demonstrate its essential role in U12 splicing mediated impact on differentiation, cell fate determination, and self-renewal potential. We will use a combination of phenotypic and in vivo and in vitro functional assays to accomplish these outcomes. By completing the proposed study, our long-term goal is to enhance our understanding of how U12 splicing regulates cellular differentiation, which will enable us to identify new drug targets and lead to robust treatment strategies for patients suffering from a growing number of diseases attributed to aberrant splicing of U12 introns.