SF3B1-targeted Splicing Inhibition Triggers Transcriptional Stress Response and Global Alterations in R-Loop Landscape

Efficient co-transcriptional splicing is thought to suppress genome-destabilizing R-loops. Inhibition of SF3B1, a core U2 spliceosome component, by Pladienolide B (PladB) in human K562 cells caused widespread intron retention and modest R-loops gains. 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 extensive 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. This resulted from increased promoter-proximal pausing and defective transcription elongation associated with premature termination. Similar results were observed upon depletion of Aquarius, a U2 spliceosome-associated factor previously thought to suppress R-loops. Thus, U2 spliceosome-targeted splicing inhibition triggered profound alterations in transcriptional dynamics, leading to unexpected disruptions in R-loop landscapes.