UC Davis

Efficient co-transcriptional splicing is thought to suppress the formation of genome-destabilizing R-loops upon interaction between nascent RNA and the DNA template. In this work, it is demonstrated that inhibition of the SF3B splicing complex using Pladienolide B (PladB) in human K562 cells caused widespread intron retention and nearly 2,000 instances of R-loops gains. However, only minimal overlap existed between these events, suggesting that unspliced introns by themselves do not cause excessive R- loops. R-loop gains were instead driven by readthrough transcription at a subset of stress-response genes, defining a new class of aberrant “downstream of genes” (DoG) R-loops. Such DoG R-loops were temporally and spatially uncoupled from loci experiencing DNA damage. Unexpectedly, the predominant response to splicing inhibition was a global R-loop loss resulting from accumulation of promoter-proximal paused RNA polymerases and defective elongation associated with premature termination. Thus, SF3B1- targeted splicing inhibition triggered profound alterations in transcriptional dynamics, leading to unexpected disruptions in the global R-loop landscape.