Wednesday, February 5, 2025
2/5/2025
Acquired mutations in splicing factors, such as SF3B1, SRSF2, and U2AF1, are prevalent in about half of all patients with the clonal myeloid disorder myelodysplastic syndromes (MDS), a disease associated with aging. Since their initial description in 2011, we have learned that these mutations are non-synonymous, mutually exclusive, and function as disease drivers. Changes in alternative splicing effected by these mutant spliceosomes have been extensively studied but are quite modest, and common patterns of splicing dysregulation that can explain the disease phenotype have not emerged. These studies have focused on the mature transcriptome (RNA-seq of polyadenylated mRNA) and discount the additional roles splicing factors play in RNA processing, such as 5’ end capping, splicing, editing and modification, 3’ end cleavage, polyadenylation, nucleocytoplasmic export, and mRNA decay and stability. Given that RNA transcription and splicing are closely coordinated (co-transcriptional splicing), we determined the changes in transcription kinetics in isogenic cells harboring the K700E mutation of SF3B1, the most mutated splicing factor in MDS. We found that elongation of RNA Polymerase II (RNAPII) leads to increased transcription-replication conflicts (TRC) and increased R-loops (three-stranded structures of newly synthesized RNA within a DNA-helix). Changes in transcription also induce changes in histone marks (H3K4me3) and chromatin organization. Using a short hairpin RNA library, we identified epigenetic regulators (in the Sin3/HDAC complex), which when knocked down reverses the transcription elongation defects and replication stress. SF3B1K700E expression also leads to a paradoxical RNA processing event: transcripts with retained introns are decreased in mutant cells compared to wild-type cells. Taken together, we hypothesize that SF3B1K700E-induced changes in RNA transcription, its subsequent processing, and chromatin organization could have disease-causing effects that are distinct from alternative splicing. The application is organized into three aims that address the following fundamental questions that arise from our preliminary data: (1) How does SF3B1K700E change RNA transcription? (2) How does SF3B1K700E impact co-transcriptional splicing and intron retention? (3) How does chromatin organization change in response to RNAPII, and how can we target them for therapeutic benefit? State-of-the art approaches to be implemented include long read sequencing (LRS) of nascent RNA to define coordination of RNA processing and splicing at the single RNA molecule level (pioneered by the Neugebauer Lab), metabolic labeling and sequencing to determine kinetics of IR-RNA decay, and multiplexed imaging of RNA (MERFISH). The results will be confirmed in primary CD34+ cultures from MDS patient samples and in a mouse model of heterozygous Sf3b1K700E mutation. Our studies will explore a novel paradigm in human disease pathogenesis: disruption of functional coupling between transcription and splicing. It will also inform ways to therapeutically target epigenetic pathways in splicing factor-mutant clonal myeloid disorders.
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