Efficient co-transcriptional splicing is thought to suppress the formation of genome-destabilizing R-loops upon interaction between nascent RNA and the DNA template. 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, arguing that unspliced introns do not cause excessive R-loops. R-loop gains were instead driven by readthrough transcription resulting from loss of transcription termination over 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. Thus, SF3B1-targeted splicing inhibition triggered profound alterations in transcriptional dynamics, leading to unexpected disruptions in the global R-loop landscape. HIGHLIGHTS • Intron retention caused by SF3B1 inhibition does not lead to excessive R-loops • A subset of genes shows readthrough transcription and accompanying R-loop gains • SF3B1 inhibition causes broad reduction in nascent transcription and R-loop loss • R-loop gains and DNA damage are temporally and spatially uncoupled RNAPII RNA +PladB PAS zZz Promoter pausing Elongation R-loops downstream of gene PAS zZz R-loops Txn read through DoG R-loops Promoter pausing Elongation No R-loop PAS zZz
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