Denaturing purifications demonstrate that PRC2 and other widely reported chromatin proteins do not appear to bind directly to RNA in vivo

Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur in vivo. Using denaturing purification of in vivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms. [Display omitted] •Many previously detected PRC2-RNA interactions do not occur in vivo•Denaturing purifications eliminate PRC2-RNA interactions but retain known interactions•Other reported chromatin regulators (CTCF and YY1) do not appear to bind RNA in vivo•Broad role for RNA binding in chromatin regulation needs to be re-evaluated This work by Guo et al. challenges widespread reports that PRC2 and other chromatin proteins bind to RNA to control the regulation of gene expression. It highlights the discrepancy between biochemical and functional evidence and suggests that reports of a broad role for protein-RNA binding in controlling chromatin regulatory mechanisms should be re-evaluated.