Rapid folding of nascent RNA regulates eukaryotic RNA biogenesis

RNA’s catalytic, regulatory, or coding potential depends on structure formation. Because base pairing occurs during transcription, early structural states can govern RNA processing events and dictate the formation of functional conformations. These co-transcriptional states remain mostly unknown. Here, we develop co-transcriptional structure tracking (CoSTseq), which detects nascent RNA base pairing within and upon exit from RNA polymerases (Pols) transcriptome wide in living yeast cells. Monitoring each nucleotide’s base pairing activity during transcription, CoSTseq reveals predominantly rapid pairing—within 25 bp of transcription after addition to the nascent chain. Moreover, ∼23% of rRNA nucleotides attain their final base pairing state near Pol I, while most other nucleotides must undergo changes in pairing status during later steps of ribosome biogenesis. We show that helicases act immediately to remodel structures across the rDNA locus to facilitate ribosome biogenesis. By contrast, nascent pre-mRNAs attain local structures indistinguishable from mature mRNAs, suggesting that refolding behind elongating ribosomes resembles co-transcriptional folding behind Pol II.