Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq) to Measure mRNA Transcription and Degradation Rates.

Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq) to Measure mRNA Transcription and Degradation Rates. Methods Mol Biol. 2020;2062:191-211 Authors: Lusser A, Gasser C, Trixl L, Piatti P, Delazer I, Rieder D, Bashin J, Riml C, Amort T, Micura R Abstract The study of RNA dynamics, specifically RNA transcription and decay rates, has gained increasing attention in recent years because various mechanisms have been discovered that affect mRNA half-life, thereby ultimately controlling protein output. Therefore, there is a need for methods enabling minimally invasive, simple and high-throughput determination of RNA stability that can be applied to determine RNA transcription and decay rates in cells and organisms. We have recently developed a protocol which we named TUC-seq to directly distinguish newly synthesized transcripts from the preexisting pool of transcripts by metabolic labeling of nascent RNAs with 4-thiouridine (4sU) followed by osmium tetroxide-mediated conversion of 4sU to cytidine (C) and direct sequencing. In contrast to other related methods (SLAM-seq, TimeLapse-seq), TUC-seq converts 4sU to a native C instead of an alkylated or otherwise modified nucleoside derivative. TUC-seq can be applied to any cell type that is amenable to 4sU labeling. By employing different labeling strategies (pulse or pulse-chase labeling), it is suitable for a broad field of applications and provides a fast and highly efficient means to dete...
Source: Mol Biol Cell - Category: Molecular Biology Authors: Tags: Methods Mol Biol Source Type: research