Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response

Abstract

Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genomewide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings.T he acute inflammatory response is critical for the defense against infections and in the healing of damaged tissues (1). The orchestration of the reprogramming of gene expression associated with the acute inflammatory response is complex and involves both transcriptional and posttranscriptional regulation (2-5). Conventional exploration of gene expression using total RNA does not fully capture this complexity because it does not provide insight into the contribution of nascent RNA synthesis or RNA decay to steady-state RNA changes. A number of different approaches have recently been developed to assess nascent RNA synthesis in cells such as global run-on and sequencing (GROSeq) (6), native elongating transcript sequencing (NET-Seq) (7), nascent RNA sequencing (Nascent-Seq) (8), and metabolic labeling of nascent RNA by using microarrays (9) or RNA-Seq (10, 11). By comparing the data obtained with metabolically labeled nascent RNA with the steady-state RNA levels, the rates of degradation of all transcripts can be computationally estimated. The stability of steady-state RNA can also be estimated from the decay rate of steady-state RNA after transcription inhibition (12)(13)(14) or by immunoprecipitation of metabolically labeled steady-state RNA after different chase periods (15, 16). These approaches work well when the system is at homeostasis, but not when conditions are altered by environmental stimuli or stress, such as the induction of the acute inflammatory response, when the rates of decay of transcripts are expected to change (10,11).In this study, we present Bru-Seq and BruChase-Seq based on bromouridine pulse labeling of nascent RNA followed by chases in uridine to obtain RNA populations of specific ages. The Brulabeled RNA is then immunocaptured followed by deep sequencing. These techniques allowed us to assess changes in the rates of both synthesis and degradation of RNA globally after the activation of the proinflammatory response by TNF. Our results provide a comprehensive and complex picture of the contribution of tr...

Large transcription units unify copy number variants and common fragile sites arising under replication stress

“…To determine whether the observations above establish a predictive model for CNV and CFS formation under replication stress, we performed a prospective study of a new cell line UMHF1 (HF1) for which we have reported detailed Bru-seq descriptions (Paulsen et al 2013b). Like 090, HF1 is a TERT-immortalized normal human fibroblast line.…”

Microprocessor dynamics shows co- and post-transcriptional processing of pri-miRNAs

“…To follow RNA from transcription through processing, actively transcribed RNA can be labeled with a pulse of a modified nucleotide (e.g., BrU) (Paulsen et al 2013) that allows for subsequent purification of RNA of a defined age during chase after stopping the labeling (Fig. 1A).…”

“…Labeled RNA was isolated as described in Paulsen et al (2013) with some modifications. Of note, 35 µL of anti-mouse IgG magnetic Dynabeads (Invitrogen) were transferred to a 1.5 mL microfuge Protein Low binding tube and washed three times with 1× BrU-IP buffer (0.1% BSA in RNAse-free PBS).…”

“…To further follow processing of pri-miRNAs endogenously over time without constraints of their cellular localization or differential transcription, we pulse-labeled RNA with bromouridine (BrU) and followed its processing during a 1-h chase (Paulsen et al 2013). We show that primiRNAs exhibit different processing kinetics both within the same polycistronic transcript and with respect to transcription and release from chromatin.…”

DRUID: a pipeline for transcriptome-wide measurements of mRNA stability

“…This approach uses nucleobase or nucleoside analogs, such as 4-thiouridine (4SU), 5-bromouridine, or 5-ethynyl uridine, all of which allow subsequent isolation of labeled RNA populations (Paulsen et al 2013;Rabani et al 2011;Tani et al 2012;Imamachi et al 2014;Neymotin et al 2014;Duffy et al 2015). The selected RNA is then quantified, often by high-throughput sequencing.…”