Comparative RNA Structure Analysis of Nascent and Mature Transcripts in Saccharomyces cerevisiae

During transcription, nascent RNA starts base pairing as it exits RNA polymerases (Pols). This base pairing permits the formation of RNA structures that critically influence gene expression at the level of RNA processing, translation, and stability. Established methods to study RNA secondary structure are limited to mature transcripts, while little is known about folding states. Moreover, the relatively lower abundance (< 1%) and transient nature of nascent RNA complicate its isolation and characterization. Co-transcriptional Structure Tracking (CoSTseq) leverages transcriptional run-on with biotin-NTP and dimethyl sulfate (DMS) probing to simultaneously acquire Pol position and base pairing status of nascent transcripts. In Saccharomyces cerevisiae, CoSTseq yields the sequence and structural information near the 3'-end of nascent RNAs transcribed by any of the three RNA Pols. During transcriptional run-on, the biotin-NTP incorporated at the active site effectively stalls Pols. Then, treatment with DMS methylates unpaired A, C, and U nucleotides. Subsequent biotin enrichment and cDNA synthesis with a template-switching reverse transcriptase enables paired-end sequencing and the computation of DMS reactivities as a function of Pol position. CoSTseq is readily performed side-by-side with DMS probing (DMS-MaPseq), enabling capture of the folded mature transcript as well. Here, a detailed protocol is presented for parallel CoSTseq and DMS-MaPseq, including transcriptional run-on, library preparation, and data analysis.