Rapid folding of nascent RNA regulates eukaryotic RNA biogenesis

An RNA’s catalytic, regulatory, or coding potential depends on RNA 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 unknown. Here, we develop CoSTseq, which detects nascent RNA base pairing within and upon exit from RNA polymerases (Pols) transcriptome-wide in living yeast cells. By monitoring each nucleotide’s base pairing activity during transcription, we identify distinct classes of behaviors. While 47% of rRNA nucleotides remain unpaired, rapid and delayed base pairing – with rates of 48.5 and 13.2 kb ^-1 of transcribed rDNA, respectively – typically completes when Pol I is only 25 bp downstream. We show that helicases act immediately to remodel structures across the rDNA locus and facilitate ribosome biogenesis. In contrast, nascent pre-mRNAs attain local structures indistinguishable from mature mRNAs, suggesting that refolding behind elongating ribosomes resembles co-transcriptional folding behind Pol II.