Correlated protein-RNA associations and a requirement for HNRNPU in long-range Polycomb recruitment 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-linked RNA-immunoprecipitations of 27 proteins from trophoblast stem cells, using a protocol exhibiting similar signal-to-noise and post-lysis reassociation ratios as CLIP and 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 degree of repression each lncRNA induced, implying connections to mechanism. Indeed, HNRNPU, an essential protein that helps Xist localize to chromatin, was enriched within and required to direct PRC-catalyzed modifications by Airn and Kcnq1ot1, without being required for their localization. Our study provides insights into the role of HNRNPU in lncRNA-mediated gene regulation and reports architectures of protein association across Airn and Kcnq1ot1 relative to the transcriptome at large. Author summaryMammalian genomes express thousands of long noncoding RNAs (lncRNAs). While most remain functionally uncharacterized, a handful are known to regulate genes via epigenetic pathways. Whether or not these known regulatory lncRNAs operate through shared or divergent mechanisms remains unclear. To gain insight, we compared RNA-protein associations across three known regulatory lncRNAs -- Airn, Kcnq1ot1, and Xist -- relative to the broader transcriptome, using a protocol to immunoprecipitate RNA from formaldehyde-crosslinked cells. Benchmarking of our RNA-immunoprecipitation protocol against methods called CLIP and CLAP revealed similar results and complementary strengths among methods. We discovered similarities in RNA-protein associations between Airn and Kcnq1ot1 that nominated them as mechanistically important. One such protein, HNRNPU, was required for Airn and Kcnq1ot1 to coordinate long-range recruitment of Polycomb complexes, without giving the appearance of being required to tether either lncRNA to chromatin, as HNRNPU has been proposed to do for Xist. Our study identifies RNA-protein associations that correlate with long-range gene regulation by lncRNAs and provides new perspectives on the potential mechanisms through which HNRNPU participates in lncRNA-mediated epigenetic control. We also benchmark a protocol to recover RNA-protein interactions that is simple to execute and has complementary strengths relative to other methods.