MicroRNAs (miRNAs) regulate the expression of most genes in animals, but we are only now beginning to understand how they are generated, assembled into functional complexes and destroyed. Various mechanisms have now been identified that regulate miRNA stability and that diversify miRNA sequences to create distinct isoforms. The production of different isoforms of individual miRNAs in specific cells and tissues may have broader implications for miRNA-mediated gene expression control. Rigorously testing the many discrepant models for how miRNAs function using quantitative biochemical measurements made in vivo and in vitro remains a major challenge for the future.

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based RNA sequencing technology that detects 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner.

In Drosophila, microRNAs (miRNAs) typically guide Argonaute1 to repress mRNA, whereas small interfering RNAs (siRNAs) guide Argonaute2 to destroy viral and transposon RNA. Unlike siRNAs, miRNAs rarely base pair extensively to the mRNAs they regulate. We find that extensive complementarity between a target RNA and an Argonaute1-bound miRNA triggers miRNA tailing and 3′-to-5′ trimming. In flies, Argonaute2-bound small RNAs—but not those bound to Argonaute1—bear a 2′-O-methyl group at their 3′ ends. This modification blocks target-directed small RNA remodeling: in flies lacking Hen1, the enzyme that adds the 2′-O-methyl group, Argonaute2-associated siRNAs are tailed and trimmed. Target-complementarity also affects small RNA stability in human cells. These results provide an explanation for the partial complementarity between animal miRNAs and their targets.