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 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. 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. 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. To investigate how nascent RNA base pairs after exit from polymerases, we developed CoSTseq. CoSTseq is a method that detects base pairing of nascent RNA. We perform CoSTseq in Saccharomyces cerevisiae of several genetic backgrounds and compared it with DMS-MaPseq of the same strains. We include at least three biological replicates for each strain.