Alternative RNA structures formed during transcription depend on elongation rate and modify RNA processing

Most RNA processing occurs co-transcriptionally. We interrogated nascent pol II transcripts by chemical and enzymatic probing and determined how the “nascent RNA structureome” relates to splicing, A-I editing and transcription speed. RNA folding within introns and steep structural transitions at splice sites are associated with efficient co-transcriptional splicing. A slow pol II mutant elicits extensive remodeling into more folded conformations with increased A-I editing. Introns that become more structured at their 3′ splice sites get co-transcriptionally excised more efficiently. Slow pol II altered folding of intronic Alu elements where cryptic splicing and intron retention are stimulated, an outcome mimicked by UV, which decelerates transcription. Slow transcription also remodeled RNA folding around alternative exons in distinct ways that predict whether skipping or inclusion is favored, even though it occurs post-transcriptionally. Hence, co-transcriptional RNA folding modulates post-transcriptional alternative splicing. In summary, the plasticity of nascent transcripts has widespread effects on RNA processing.