Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry.
We describe a chemical method to label and purify 4-thiouridine (s(4)U)-containing RNA. We demonstrate that methanethiosulfonate (MTS) reagents form disulfide bonds with s(4)U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s(4)U 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.
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA.
Erin E
EE
Duffy
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA.
Michael
M
Rutenberg-Schoenberg
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA.
Catherine D
CD
Stark
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA.
Robert R
RR
Kitchen
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA.
Mark B
MB
Gerstein
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA. Electronic address: matthew.simon@yale.edu.
Matthew D
MD
Simon
D001710:Biotin / Q000031:analogs & derivatives; D049109:Cell Proliferation; D004220:Disulfides; D020869:Gene Expression Profiling / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D007202:Indicators and Reagents; D008698:Mesylates; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055601:Organic Chemistry Phenomena; D012323:RNA Processing, Post-Transcriptional; D013891:Thiouridine / Q000737:chemistry
D004220:Disulfides; D007202:Indicators and Reagents; D008698:Mesylates; D035683:MicroRNAs; C501660:N-(6-(biotinamido)hexyl)-3'-(2'-pyridyldithio)propionamide; D013891:Thiouridine; C074502:methanethiosulfonate; D001710:Biotin