The varying rates at which mRNAs decay are tightly coordinated with transcriptional changes to shape gene expression during development and disease. But currently available RNA sequencing approaches lack the temporal information to determine the relative contribution of RNA biogenesis, processing and turnover to the establishment of steady-state gene expression profiles.

Here, we describe a protocol that combines metabolic RNA labeling with chemical nucleoside conversion by thiol-linked alkylation of 4-thiouridine to determine RNA stability in cultured cells (SLAMseq). When coupled to cost-effective mRNA 3′ end sequencing approaches, SLAMseq determines the half-life of polyadenylated transcripts in a global and transcript-specific manner using untargeted or targeted cDNA library preparation protocols.

We provide a step-by-step instruction for time-resolved mRNA 3′ end sequencing, which augments traditional RNA-seq approaches to acquire the temporal resolution necessary to study the molecular principles that control gene expression.

Features SLAMseq Metabolic RNA Labeling Kit for RNA-Seq, QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina and QuantSeq-Flex Targeted RNA-Seq Library Prep Kit V2 for Illumina

The posttranscriptional addition of nucleotides to the 3′ end of RNA regulates the maturation, function, and stability of RNA species in all domains of life. Here, we show that in flies, 3′ terminal RNA uridylation triggers the processive, 3′‐to‐5′ exoribonucleolytic decay via the RNase II/R enzyme CG16940, a homolog of the human Perlman syndrome exoribonuclease Dis3l2. Together with the TUTase Tailor, dmDis3l2 forms the cytoplasmic, terminal RNA uridylation‐mediated processing (TRUMP) complex that functionally cooperates in the degradation of structured RNA. RNA immunoprecipitation and high‐throughput sequencing reveals a variety of TRUMP complex substrates, including abundant non‐coding RNA, such as 5S rRNA, tRNA, snRNA, snoRNA, and the essential RNase MRP. Based on genetic and biochemical evidence, we propose a key function of the TRUMP complex in the cytoplasmic quality control of RNA polymerase III transcripts. Together with high‐throughput biochemical characterization of dmDis3l2 and bacterial RNase R, our results imply a conserved molecular function of RNase II/R enzymes as “readers” of destabilizing posttranscriptional marks—uridylation in eukaryotes and adenylation in prokaryotes—that play important roles in RNA surveillance.

Features QuantSeq-Flex Targeted RNA-Seq Library Prep Kit