Gaining insight into transcriptome‐wide RNA population dynamics through the chemistry of 4‐thiouridine

Cellular RNA levels are the result of a juggling act between RNA transcription, processing, and degradation. By tuning one or more of these parameters, cells can rapidly alter the available pool of transcripts in response to stimuli. While RNA sequencing (RNA-seq) is a vital method to quantify RNA levels genome-wide, it is unable to capture the dynamics of different RNA populations at steady-state or distinguish between different mechanisms that induce changes to the steady-state (i.e. altered rate of transcription versus degradation). The dynamics of different RNA populations can be studied by targeted incorporation of non-canonical nucleosides. 4-thiouridine (s4U) is a commonly used and versatile RNA metabolic label that allows the study of many properties of RNA metabolism from synthesis to degradation. Numerous experimental strategies have been developed that leverage the power of s4U to label newly transcribed RNA in whole cells, followed by enrichment with activated disulfides or chemistry to induce C mutations at sites of s4U during sequencing. This review presents existing methods to study RNA population dynamics genome-wide using s4U metabolic labeling, as well as a discussion of considerations and challenges when designing s4U metabolic labeling experiments.