title: Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability.
abstract: Changes in the rate of mRNA decay are closely coordinated with transcriptional changes and together these events have profound effects on gene expression during development and disease. Traditional approaches to assess mRNA decay have relied on inhibition of transcription, which can alter mRNA decay rates and confound interpretation. More recently, metabolic labeling combined with chemical modification and fractionation of labeled RNAs has allowed the isolation of nascent transcripts and the subsequent calculation of mRNA decay rates. This approach has been widely adopted for measuring mRNA half-lives on a global scale, but has proven challenging to use for analysis of single genes. We present a series of normalization and quality assurance steps to be used in combination with 4-thiouridine pulse labeling of cultured eukaryotic cells. Importantly, we demonstrate how the relative amount of 4sU-labeled nascent RNA influences accurate quantification. The approach described facilitates reproducible measurement of individual mRNA half-lives using 4-thiouridine and could be adapted for use with other nucleoside analogs.
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80525, United States.
authors.forename: Joseph
authors.initials: J
authors.lastname: Russo
authors.affiliation: Program in Cell & Molecular Biology, Colorado State University, Fort Collins, CO 80525, United States.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Heck
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80525, United States; Program in Cell & Molecular Biology, Colorado State University, Fort Collins, CO 80525, United States. Electronic address: jeffrey.wilusz@colostate.edu.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Wilusz
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80525, United States; Program in Cell & Molecular Biology, Colorado State University, Fort Collins, CO 80525, United States.
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Wilusz
mesh_terms: D000345:Affinity Labels / Q000737:chemistry*; D000818:Animals; D019921:Biotinylation / Q000379:methods; D002460:Cell Line; D005057:Eukaryotic Cells / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D051379:Mice; D011786:Quality Control; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*; D013891:Thiouridine / Q000737:chemistry*; D014158:Transcription, Genetic
chemical_list: D000345:Affinity Labels; D012333:RNA, Messenger; D013891:Thiouridine
keywords: 4-Thiouridine labeling; Digital PCR; RNA biotinylation; mRNA decay; mRNA stability