Correlated protein-RNA associations and a requirement for HNRNPU in the long-range recruitment of Polycomb Repressive Complexes by the lncRNAs Airn and Kcnq1ot1.

The lncRNAs Airn and Kcnq1ot1 recruit Polycomb Repressive Complexes (PRCs) and repress genes over multi-megabase genomic intervals, but how they interact with proteins to direct repression remains poorly understood. We conducted formaldehyde-based RNA-immunoprecipitations (RIPs) of 27 proteins from mouse trophoblast stem cells (TSCs), using a protocol exhibiting similar signal-to-non-specific signal and post-lysis reassociation ratios as crosslinking immunoprecipitation (CLIP) and crosslinking affinity purification (CLAP). Patterns of protein associations across Airn and Kcnq1ot1 were more similar to each other than to nearly all other transcripts and partitioned to extents that mirrored the degree of repression each lncRNA induced, implying connections to mechanism. Indeed, HNRNPU, a factor essential for Xist's localization to chromatin, was enriched over Airn and Kcnq1ot1 and required to maintain normal levels of PRC1- and PRC2-directed chromatin modifications across the Airn and Kcnq1ot1 target domains, yet was dispensable for both lncRNAs' localization to chromatin and for their association with PRC1. HNRNPU depletion caused a greater reduction in PRC-directed chromatin modifications and gene repression across the inactive X and the ~ 15 Mb Airn target domain than across the ~ 3 Mb Kcnq1ot1 domain. Perhaps relatedly, HNRNPU depletion significantly reduced the overall levels of Xist and Airn but not Kcnq1ot1. Our study reports architectures of protein association along Airn and Kcnq1ot1 compared to the transcriptome at large, highlights shared and distinct features between the two lncRNAs, and provides new perspective on the role of HNRNPU in long-range chromatin regulation by lncRNAs.