Rapid and scalable profiling of nascent RNA with fastGRO

Abstract

Genome-wide profiling of nascent RNA has become a fundamental tool to study transcription regulation. Over the past decade, next-generation sequencing has fostered development of a handful of techniques (i.e. GRO-seq, PRO-seq, TT-seq and NET-seq) that map unprocessed transcripts originating from both the coding and the noncoding portion of the genome. Unlike steady-state RNA sequencing, nascent RNA profiling mirrors the real-time activity of RNA Polymerases and provides an accurate readout of transcriptome-wide variations that occur during short time frames (i.e. response to external stimuli or rapid metabolic changes). Some species of nuclear RNAs, albeit functional, have a short half-life and can only be accurately gauged by nascent RNA techniques (i.e. lincRNAs and eRNAs). Furthermore, these techniques capture uncapped post-cleavage RNA at termination sites or promoter-associated antisense RNAs, providing a unique insight into RNAPII dynamics and processivity.Here we present a run-on assay with 4s-UTP labelling, followed by reversible biotinylation and affinity purification via streptavidin. Our protocol allows streamlined sample preparation within less than 3 days. We named the technique fastGRO (fast Global Run-On). We show that fastGRO is highly reproducible and yields a more complete and extensive coverage of nascent RNA than comparable techniques. Importantly, we demonstrate that fastGRO is scalable and can be performed with as few as 0.5x10^6 cells.

A PP2A-Integrator complex fine-tunes transcription by opposing CDK9

“…To determine the effect of INTS6/PP2A loss on acutely activated genes that were suppressed by CDK9i, INTS6 was depleted in HeLa cells ( Figure S5J) and cells were pre-treated with AZ5576 for two hours prior to stimulation with EGF for 15 minutes. ChIP-seq assays demonstrated that total RNAPII and RNAPII phospho-Ser2 accumulated genome-wide in INTS6-depleted HeLa cells treated with AZ5576, with accumulation most clearly observed across the gene bodies of EGF-responsive genes, in contrast to the reduced ChIP-seq signal observed in control shLUC cells ( Figure 5H Consistent with the ChIP-seq data for RNAPII and RNAPII phospho-Ser2 detailed above, fastGRO assays (Barbieri et al, 2020) to assess nascent RNA production demonstrated that depletion of INTS6 in HeLa cells counteracted CDK9i-induced transcriptional pausing at all EGF-stimulated genes ( Figure 5J-K). A broader analysis of the highest-expressed genes (n=2989) demonstrated that INTS6 depletion decreased the pausing index of nearly all active genes ( Figure 5L), similar to the effects observed in THP-1 sgINTS6-KO cells treated with AZ5576 ( Fig 5F).…”

“…FastGRO was performed as described (Barbieri et al, 2020). HeLa cells infected with shLUC or shINTS6 were treated with DMSO or 300nM CDK9i AZ5576 for 2 hours, then subsequently treated for 15 minutes with 0.1μg/mL EGF.…”

The PP2A-Integrator-CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer

“…In agreement with a role for INTS8 in PP2A recruitment, its depletion phenocopied INTS6 loss and conferred resistance to CDK9i-induced pausing (Figures S5O-S5Q). FastGRO assays (Barbieri et al, 2020) measuring nascent RNA production also showed that INTS6 depletion counteracted CDK9i-induced transcriptional pausing at all EGF-stimulated genes, whereas a broader analysis of the most highly expressed genes demonstrated that INTS6 depletion decreased the pausing index at these active loci (Figures 5J-5L). To determine whether PP2A depletion can also drive escape from CDK9i-induced pausing, analogous assays were also performed in HeLa cells with shRNA-mediated knockdown of PPP2R1A (Figure S5R).…”

“…FastGRO was performed as described (Barbieri et al, 2020). HeLa cells infected with shLUC or shINTS6 were treated, 72 hours after infection, with DMSO or 300nM CDK9i AZ5576 for 2 hours, then subsequently treated for 15 minutes with 0.1 mg/mL EGF.…”

Protocol Variations in Run-On Transcription Dataset Preparation Produce Detectable Signatures in Sequencing Libraries

“…To capture all RNAs arising from cellular RNA polymerases, several run-on transcription capture protocols, such as global run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq), have been developed [1,8,3,9,10]. These protocols, collectively known as RO-seq, follow roughly a two step process: first, the run-on RNA signal must be enriched above the background total RNA; second, the captured RNA must then be converted into a sequencing-ready cDNA library [1].…”