Impact of Methods on the Measurement of mRNA Turnover

Abstract

The turnover of the RNA molecules is determined by the rates of transcription and RNA degradation. Several methods have been developed to study RNA turnover since the beginnings of molecular biology. Here we summarize the main methods to measure RNA half-life: transcription inhibition, gene control, and metabolic labelling. These methods were used to detect the cellular activity of the mRNAs degradation machinery, including the exo-ribonuclease Xrn1 and the exosome. On the other hand, the study of the differential stability of mature RNAs has been hampered by the fact that different methods have often yielded inconsistent results. Recent advances in the systematic comparison of different method variants in yeast have permitted the identification of the least invasive methodologies that reflect half-lives the most faithfully, which is expected to open the way for a consistent quantitative analysis of the determinants of mRNA stability.

Characterizing RNA stability genome-wide through combined analysis of PRO-seq and RNA-seq data

“…We found that the two sets of estimates were reasonably well correlated (Spearman's ρ=0.54; Fig. 2B), especially considering the substantial differences in experimental protocols and the generally poor concordance of published half-life estimates across experimental methods 6,15 . Moreover, if we remove the 50% of genes expressed at the lowest levels (as measured by PRO-seq), for which the noise contribution will tend to be largest, the correlation improves to ρ=0.61.…”

“…Over a period of more than four decades, investigators have developed numerous methods for measuring RNA decay rates or half-lives [13][14][15] . A classical approach to this problem, still in use today, is to measure the decay in RNA abundance over time following inhibition of transcription, often using the antibiotic actinomycin D 1,3,16 .…”

“…More recently, many studies have employed a strategy that is conceptually similar but considerably less disruptive to cellular physiology, based on metabolic labeling of RNA transcripts with modified nucleotides. In this approach, the relative proportions of labeled and unlabeled transcripts are quantified as they change over time due to RNA turnover, following an initial introduction or removal of labeled nucleotides 6,15 . Today, metabolic labeling is most commonly accomplished using the nucleotide analog 4-thiouridine (4sU, also known as s 4 U), which is rapidly taken up by animal cells and can be biotinylated for affinity purification 4,7,9,[17][18][19] .…”

“…Many of these methods also have limited sensitivity for low-abundance transcripts. Owing to various limitations of the assays, differences in the populations of accessible TUs, and potential disruptions to cellular physiology, estimates of RNA half-lives tend to vary considerably across assays, with median half-lives often differing by factors of 2-3 or more 6,15 . As yet, there exists no general-purpose assay for RNA half-life that is as robust, sensitive, convenient, or versatile as RNA-seq is for measuring cellular RNA concentrations, or PRO-seq 21 and NET-seq 22 are for measuring nascent transcription.…”

Identification and dynamic quantification of regulatory elements using total RNA

“…RNA stability presents a continuum spanning RNA half-lives of less than a minute to several hours, which impacts their detection (Wada and Becskei 2017). Unstable RNAs and their initiation sites are difficult to identify with methods that capture steady-state RNA levels such as conventional RNA-seq or 5 ′ 7-methylguanosine cap (5 ′ cap)-enriched RNA sequencing methods such as 5 ′ RNA-seq or CAGE (Shiraki et al 2003).…”