thiouridine and eniluracil

Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. This review focuses on the methodology used to specifically measure RNA decay at a global level. By comparing and contrasting approaches and describing the experimental protocols in a modular manner, we intend to provide both experienced and new researchers to the field the ability to combine aspects of various protocols to fit the unique needs of biological questions not addressed by current methods.

Topics: Animals; Biotin; Bromouracil; Cell Line; Click Chemistry; High-Throughput Nucleotide Sequencing; Humans; RNA Stability; RNA, Messenger; Staining and Labeling; Thiouracil; Thiouridine; Transcriptome; Uracil; Uridine

Changing the abundance of transcripts by regulated RNA degradation is a critical step in the control of various biological pathways. Recently, genome-wide inhibitor-free technologies for determining RNA stabilities in mammalian cells have been developed. In these methods, endogenous RNAs are pulse labeled by uridine analogs [e.g., 4-thiouridine (4sU), 5-etyniluridine (EU) and 5'-bromo-uridine (BrU)], followed by purification of labeled de novo RNAs. These technologies have revealed that the specific half-life of each mRNA is closely related to its physiological function. Genes with short-lived mRNAs are significantly enriched among regulatory genes, while genes with long-lived mRNAs are enriched among housekeeping genes. This review describes the recent progress of experimental procedures for measuring RNA stability.

Topics: Alpha-Amanitin; Animals; Bromouracil; Dactinomycin; Eukaryotic Cells; Genes, Essential; Genes, Regulator; Half-Life; Humans; Nucleic Acid Synthesis Inhibitors; RNA Stability; RNA, Messenger; Thiouridine; Transcription, Genetic; Uracil; Uridine