In Vitro Transcription Recycling Assay Identifies PAF1 as a Driver of RNA Pol II Recycling

RNA Polymerase II transcriptional recycling is an underappreciated mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of novel RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from RNA Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed in vivo PAF1 complex cycling through gene bodies with RNA Pol II and recycling back to the transcription start sites, while PAF1 silencing triggered defective RNA Pol II recycling. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify RNA Pol II recycling as a potential target in cancer and demonstrate the assay's applicability to characterize RNA Pol II recycling in cells and tissues from other disease states. Overall design: We have performed time-resolved ChIP-seq of PAF1 and Pol II. PAF1 and Pol II ChIP-seq time course was generated during 0-, 10-, 20-, or 40-minute Flavopiridol treatment to block release of new Pol II into gene bodies and during 0-, 10-, 20- or 40-minute recovery time points after wash-out of Flavopiridol to release Pol II into the gene bodies in LNCaP-abl cells transfected with either non-targeting control siRNA or siPAF1.