Determining mRNA half-lives on a transcriptome-wide scale

Abstract

Every step in the life cycle of an RNA transcript provides opportunity for regulation. One important aspect of post-transcriptional control is the regulation of RNA stability. Of the many strategies for determining mRNA stability, transcription inhibition and metabolic labeling have proved the most amenable to high-throughput analysis and have opened the door to dissecting mRNA decay transcriptome-wide. Here, we describe experimental and computational methods to determine transcriptome-wide RNA stabilities using both pharmacological inhibition of transcription and metabolic labeling. To aid in the analysis of these experiments, we discuss key characteristics of high-quality experiments and address other experimental and computational considerations for the analysis of mRNA stability. Broader application of these approaches will further our understanding of mRNA decay and illuminate its contribution to different biological processes.

Differential contribution of steady‐state RNA and active transcription in chromatin organization

“…Previous studies have provided evidence that the act of transcription may play a role in shaping chromatin organization [26,27,29,30,34,35], yet the global consequences of complete transcriptional arrest (i.e., blocking all three polymerases) on mammalian chromosomal architecture are not well understood. Accordingly, to assess whether genome organization is affected differently by the act of transcription compared to loss of steady-state RNA following RNase treatment, we treated K562 cells with actinomycin D (ActD), a drug that quickly (i.e., within 24 h) and irreversibly inhibits all polymerases and results in complete transcriptional arrest [36][37][38] ( Fig 1C). Agarose gel electrophoresis analysis of total RNA extracted from the same number of cells as controls upon 24 h of ActD treatment revealed that~50% of the 28S/18S ribosomal RNA bands remain, indicating the inhibition of global transcription and a decrease in nuclear RNA content ( Fig 1D).…”

Short residence times of DNA-bound transcription factors can reduce gene expression noise and increase the transmission of information in a gene regulation system

“…For mRNA, experimentally measured half-lives usually lie in the range of seconds to hours (61)(62)(63)(64). Protein half-lives are usually longer than mRNA half-lives (65) and lie between hours and days (63,66).…”

Transcriptome stability profiling using 5’-bromouridine IP chase (BRIC-seq) identifies novel and functional microRNA targets in human melanoma cells

“…However, RNA-seq represents a static image of the process of transcription, while biological processes are by their very nature dynamic. Recent studies on transcriptome dynamics have shown that transcript stability is correlated with function [3][4][5][6]. For instance, transcription factor mRNAs have high average decay rates [6] and mRNA turnover correlates with signaling pathways active in cancer, suggesting a post-transcriptional component to oncogenesis [7].…”

“…Hence, understanding transcriptional dynamics is important for understanding disease pathogenesis including in cancer. mRNA decay studies have traditionally been dominated by the use of transcriptional inhibitors such as actinomycin D [8,4] -amanitin [3,9] to arrest transcription. mRNA concentrations are then quantified at progressive time points after transcriptional arrest.…”

“…However, these transcriptional inhibitors stress cells and themselves alter transcript localization, stability, and cellular proliferation, introducing unintended and confounding variables [10,11]. More recent metabolic nucleotide labeling techniques such as with 5bromouridine [12,10], 4-thiouridine [3,10], or 5-ethynyluridine [13] have overcome these limitations by providing a direct read-out of transcript decay. Here -bromouridine IP chase (BRIC)-seq assay [12,10], in which the nucleotides of newly synthesized transcripts are labeled with 5-bromouridine (BrU), to study transcriptomic stability in melanoma cells.…”