This Title All WIREs
How to cite this WIREs title:
Impact Factor: 4.928

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

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

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 vs. degradation). The dynamics of different RNA populations can be studied by targeted incorporation of noncanonical 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. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Turnover and Surveillance > Regulation of RNA Stability
(a) Overview of 4‐thiouridine (s4U) metabolic labeling experiments. Different options are indicated for each module, along with references for representative examples. In the case where two references are listed, the first applies to yeast and the second to mammalian cells, as the time of s4U labeling can vary widely between organisms. (b) Representative examples of two s4U metabolic labeling experiments that leverage different module options: 4sUDRB‐seq to measure RNA polymerase II elongation and TT‐TimeLapse‐seq to detect unstable RNAs. RT‐qPCR = quantitative reverse transcription PCR; DRB = 5,6‐dichloro‐1‐β‐D‐ribofuranosyl‐1H‐benzimidazole
[ Normal View | Magnified View ]
(a) Hydrogen bonding pattern of modified or recoded 4‐thiouridine (s4U) nucleosides following enrichment‐free chemistry. (b) Example, TimeLapse‐seq tracks (data from Schofield et al., ) for faster turnover (Ier3) and slower turnover (Srsf3) transcripts in MEF cells treated with 1 mM s4U for 1 hr
[ Normal View | Magnified View ]
Methods to normalize 4‐thiouridine (s4U)‐RNA data after high‐throughput sequencing. (a) After cells are metabolically labeled with s4U and total RNA is extracted, exogenous s4U‐RNA (s4U‐labeled RNA from S. pombe or in vitro‐transcribed s4U‐RNAs) are added to each sample and then enriched. Alternatively or in addition, exogenous RNA without s4U can also be added to samples after enrichment. Samples are then analyzed by high‐throughput sequencing and normalized based on the number of reads that align to the spike‐in sample. (b) Samples can also be enriched without RNA spike‐ins and normalized based on the relative proportion of introns in each sample (Lugowski et al., ). First, intron coverage for each transcript is determined based on the number of reads that map to introns in the longest isoform of a given transcript without overlapping exons or any other isoform. Next, introns are filtered for coverage and time‐dependent changes in expression. Reads that map to exons are finally normalized to the sum of all reads mapping to the well‐behaved intron set
[ Normal View | Magnified View ]
Significance of inefficient chemistry toward length bias in 4‐thiouridine (s4U) metabolic labeling experiments. (a) Chemistry of activated disulfide reactivity with s4U. When HPDP‐biotin is used as the activated disulfide, any biotin‐s4U (bio‐s4U) product results in a more activated disulfide due to the electron‐poor pyrimidine ring of s4U, therefore, favoring the reverse rather than the forward biotinylation reaction. In contrast, MTS‐biotin reacts irreversibly with s4U to form the bio‐s4U product under the reaction conditions, and the covalent disulfide bond can only be reversed under reducing conditions. (b) Schematic diagram of s4U‐RNA enrichment with HPDP‐ and MTS‐biotin. Efficient activated disulfide reactivity of MTS‐biotin results in greater yields of s4U‐RNA and alleviates potential biases toward longer RNAs with more uridines
[ Normal View | Magnified View ]
(a) Estimated range of RNA half‐lives in mammalian cells based on data from Schofield et al. (), Schwalb et al. (), and Yang et al. (). (b) Simulation of RNA decay rates for 4‐thiouridine (s4U) pulse‐chase experiment for three different RNA half‐lives, represented as a fraction of the total amount of s4U‐labeled RNA at the start of the chase. (c) Simulation of s4U approach‐to‐equilibrium kinetics for three different RNA half‐lives. (d) Kinetics of s4U incorporation during 5 min of s4U labeling for a transient RNA (t1/2 = 5 min) and two mRNAs (t1/2 = 0.5 and 4 hr)
[ Normal View | Magnified View ]

Related Articles

Top Ten WRNA Articles

Browse by Topic

RNA Turnover and Surveillance > Regulation of RNA Stability
RNA Methods > RNA Analyses in Cells

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

The latest WIREs articles in your inbox

Sign Up for Article Alerts