Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry

Abstract

We describe a chemical method to label and purify 4-thiouridine (s4U)-containing RNA. We demonstrate that methanethiosulfonate (MTS) reagents form disulfide bonds with s4U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s4U labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA, such as 4-thiouridine tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover. Duffy et al. demonstrate that previously used chemistry to enrich 4-thiouridine-containing RNA (s4U-RNA) is inefficient and produces biased results, whereas their MTS chemistry allows for efficient s4U-RNA labeling and enrichment. The authors use this chemistry to improve s4U-based metabolic labeling experiments and study the turnover of microRNAs.