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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Nascent RNA folding during transcription influences gene expression.
• Existing methods primarily study mature RNA structures, not transient nascent states.
• Low abundance and transient nature of nascent RNA hinder its study.

Purpose of the Study:

• To present a detailed protocol for Co-transcriptional Structure Tracking (CoSTseq).
• To enable simultaneous analysis of nascent RNA structure and polymerase position.
• To facilitate the study of RNA folding states during transcription.

Main Methods:

• CoSTseq utilizes transcriptional run-on with biotin-NTP to stall polymerases.
• Dimethyl sulfate (DMS) probing methylates unpaired nucleotides in nascent RNA.
• Biotin enrichment, cDNA synthesis, and sequencing provide structural and positional data.

Main Results:

• CoSTseq provides sequence and structural information for nascent RNAs in Saccharomyces cerevisiae.
• The method captures RNA folding near the 3'-end of transcripts from all three RNA polymerases.
• CoSTseq can be performed in parallel with DMS-MaPseq to study mature transcripts.

Conclusions:

• CoSTseq offers a powerful approach to study nascent RNA structures in vivo.
• This technique enhances understanding of co-transcriptional RNA folding and gene regulation.
• The protocol facilitates parallel analysis of nascent and mature RNA structures.