thiouridine and methanethiosulfonate

Here, we describe an approach to enrich newly transcribed RNAs from primary mouse neurons using 4-thiouridine (s4U) metabolic labeling and solid phase chemistry. This one-step enrichment procedure captures s4U-RNA by using highly efficient methane thiosulfonate (MTS) chemistry in an immobilized format. Like solution-based methods, this solid-phase enrichment can distinguish mature RNAs (mRNA) with differential stability, and can be used to reveal transient RNAs such as enhancer RNAs (eRNAs) and primary microRNAs (pri-miRNAs) from short metabolic labeling. Most importantly, the efficiency of this solid-phase chemistry made possible the first large scale measurements of RNA polymerase II (RNAPII) elongation rates in mouse cortical neurons. Thus, our approach provides the means to study regulation of RNA metabolism in specific tissue contexts as a means to better understand gene expression in vivo.

Topics: Animals; Cell Line, Tumor; Gene Expression; HEK293 Cells; Humans; Mesylates; Mice; MicroRNAs; Neurons; RNA; RNA Polymerase II; Staining and Labeling; Thiouridine

Metabolic labeling of cellular RNA is a useful approach to study RNA biology. 4-Thiouridine (s

Topics: Biotin; HEK293 Cells; Humans; Mesylates; RNA; Streptavidin; Thiouridine

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.

Topics: Biotin; Cell Proliferation; Disulfides; Gene Expression Profiling; HEK293 Cells; Humans; Indicators and Reagents; Mesylates; MicroRNAs; Organic Chemistry Phenomena; RNA Processing, Post-Transcriptional; Thiouridine