Splicing Inhibition Reveals Novel Roles for SF3B1 in Transcription Dynamics and R-Loop Metabolism

Efficient co-transcriptional splicing has been proposed to suppress the formation of genome-destabilizing R-loops upon interaction between nascent RNA and the DNA template. To test this, we inhibited the SF3B splicing complex using Pladienolide B (PladB) in human K562 cells and mapped R-loop genomic distributions. PladB caused widespread intron retention, as expected, and nearly 2,000 instances of R-loops gains. However, only minimal overlap existed between these two events, arguing that unspliced introns do not cause excessive R-loops. Instead, R-loop gains were driven by a loss of transcription termination over a specific subset of stress-response genes, defining a new class of “downstream of genes” (DoG) aberrant R-loops. Unexpectedly, the predominant response to splicing inhibition was a global R-loop loss over thousands of genes, resulting from a profound loss of transcription elongation. Thus, acute splicing inhibition triggered profound and contrasting alterations in transcriptional dynamics, which were reflected in the global R-loop landscape. Overall design: RNA-seq, EU-seq, DRIP-seq, and sDRIP-seq were performed on control or treated K562 cells to characterize their polyA RNA, nascent RNA, and R-loop profiles. Specifically, K562 cells were subjected to either nothing, DMSO, or Pladienolide B; each for 2 hours and 4 hours. Each experiment was performed with at least 2 biological replicates.