Establishing RNA-RNA interactions remodels lncRNA structure and promotes PRC2 activity

Abstract

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

Fig. 1. HOTAIR intermolecular interaction is promoted by hnRNP B1.

(A) Model of B1-mediated HOTAIR RNA-RNA interactions with nascent target RNA leading to PRC2 activity and gene silencing. (B) In vitro RNA pulldown with MS2-tagged HOTAIR or Anti-luc with recombinant B1 or A2. “A2+” is 3× the concentration of A2. Minus MS2-MBP fusion protein pulldown was included to account for background bead binding. Western blot analysis was performed using A2/B1 antibody. RNA recovery was quantified by quantitative polymerase chain reaction (qPCR; n = 3). (C) Crystal structure of RRM domains of A2/B1 in complex with 10-mer RNA (yellow) (27). Two molecules of the tandem RRMs are shown in purple/green. Blue circles highlight the N terminus. Adapted from (27) (http://creativecommons.org/licenses/by/4.0/). (D) Schematic of RNA-RNA interaction assay: RAT-tagged JAM2 and HOTAIR in vitro transcriptions (IVTs) incubated ± recombinant hnRNP B1. JAM2 tethered by PP7 coat protein on magnetic beads. Recovery of HOTAIR by JAM2 pulldown was quantified by RT-qPCR and protein by Western blot. (E) Assays from (D) with HOTAIR or Anti-luc RNA ± hnRNP B1 or A2 (n = 6). (F) As in (E) with full-length HOTAIR, HOTAIR with the JAM2 interaction site deleted or mutated ± hnRNP B1 (n = 3). Error bars in (E) and (F) represent SDs. P values were determined using two-way analysis of variance (ANOVA) and two-tailed Student’s t tests. n.s., not significant; WT, wild type; Pol II, RNA Polymerase II.

Fig. 2. Examining hnRNP B1–specific interactions with the lncRNA HOTAIR using in vitro eCLIP mapping.

(A) Schematic of in vitro eCLIP experiments: recombinant B1 incubated with IVT HOTAIR. HOTAIR-B1 complexes were UV–cross-linked. RNA was fragmented with limited RNase A treatment, followed by in vitro eCLIP protocol [see Materials and Methods and (24)]. (B) Top: Mapping of RT termination events from HOTAIR-B1 in vitro eCLIP as a measure of direct protein cross-linking (HOTAIR domains alternately shaded violet). Termination sites normalized to read count with significant peaks were determined by values greater than 5000. Bottom: Zoom-in on domain 1 (1 to 530) for titration experiments highlights multiple B1 interaction sites (shaded in gray). (C) Top: Diagram of constructs including construct with all RGGs mutated (“5XRGG MUT”) and B1 glycine-rich domain deletion (“ΔGR”). Bottom: Assays as in Fig. 1B with recombinant truncated versions of the constructs depicted above. Western blot for A2/B1. Intensities should be compared to input, since the antibody recognizes the constructs differentially. Equal protein loading for samples is demonstrated by Coomassie gel with equal amounts of protein loaded as in each pulldown. Bar graph of HOTAIR recovery as percent input from each pulldown was quantified by qPCR (n = 2).

Fig. 3. Chemical probing of HOTAIR highlights B1 interactions.

(A) Diagram of the IVT HOTAIR domain 1 construct for chemical probing experiments. Schematic representation of 1M7 chemical probing of HOTAIR domain 1 with B1 and/or the JAM2 fragment (54 nt). 5′ SL, 5′ stem-loop. (B) Heatmap of normalized reactivity for HOTAIR only (HA only), HOTAIR + JAM2 (HAJ2), HOTAIR + B1 (HAB1), and HOTAIR + B1 + JAM2 (HAB1J2). White values represent nonreactive nucleotides, and red values represent more reactive nucleotides. (C) Line graphs of normalized reactivity for each condition. Light blue shaded boxes highlight the in vitro B1 eCLIP sites identified. Light red shaded box highlights the RNA-RNA interaction site. (D) Boxplots of normalized reactivity for all nucleotide positions, the JAM2 interaction region (245 to 306 nt), and B1 eCLIP-derived binding sites (141 to 172, 304 to 314, and 460 to 523 nt). Error bars represent SDs. P values were determined using one-way ANOVA multiple comparison tests between HA only compared to each variable condition (*P < 0.01, **P < 0.005, and ***P < 0.0001). (E) Bar graphs for normalized reactivity of HOTAIR only and HOTAIR + B1 at specific eCLIP B1 binding sites, as well as a minimally changed control region, by nucleotide.

Fig. 4. Establishment of RNA-RNA interactions alters HOTAIR structure.

(A) Heatmap of HOTAIR reactivity changes upon addition of JAM2 (ΔJ2), hnRNP B1 (ΔB1), or both (ΔB1J2), compared to HOTAIR only. Red values become more reactive, white values do not change, and blue values become less reactive when JAM2, B1, or both are present. (B) Prominent regions of change mapped to the HOTAIR domain 1 secondary structure (28) (with permission) with either JAM2 RNA, (C) hnRNP B1 (highlighted in gray), or (D and E) both JAM2 + B1 using either control RNA threshold analysis (highlighted in light green) or control protein threshold analysis (highlighted in light purple). Regions of change were determined as described in Materials and Methods. In vitro eCLIP B1 binding sites are highlighted in blue, and the JAM2 RNA-RNA interaction site is highlighted in green.

Fig. 5. Duplex RNA promotes PRC2 activity.

(A) Histograms comparing the predicted minimum free energy for RNA-RNA interactions between HOTAIR and 40,740 RNAs across the transcriptome (gray) or between HOTAIR and 885 transcripts from genes that gain PRC2 activity when HOTAIR is overexpressed in breast cancer cells (red). Data are from (23, 25, 38). (B) Native gel of dinucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40 bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. (C) Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4, and AEBP2, analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie blue. (D) HMTase assay was performed with recombinant PRC2 complex, hexanucleosomes, S-adenosylmethionine with and without the cofactor JARID2 (amino acids 119 to 574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. (E) Native 0.5× tris-borate-EDTA gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration analyzed by Western blot.

Fig. 6. HOTAIR matching with target RNA promotes PRC2 activity.

(A) Native PAGE of RNA annealing titration with HOTAIR fragments and either JAM2 or HOXD10 matching RNAs. (B) RNA-RNA interaction between HOTAIR and JAM2 predicted by IntaRNA (50). HMTase assay performed with recombinant PRC2, dinucleosomes, HOTAIR fragment (62 nt), and JAM2 match (54 nt) titration. PRC2 activity was determined by H3K27me3 and total H3 by Western blot. (C) Quantification of (B) (n = 3). (D) IntaRNA result of HOTAIR and HOXD10. HMTase as in (B) with HOTAIR fragment (31 nt) HOXD10 match (37 nt) titration. (E) Quantification of (D) (n = 5). (F) As in (D), with poly(A) instead of HOXD10 (n = 4). (G) HMTase as above with HOTAIR domain 1 (nucleotides 1 to 530) and JAM2 match (62 nt) titration. (H) Quantification of (G) (n = 3). (I) As in (G), except using HOTAIR with JAM2 match site deleted “HOTAIR D1_del.” (J) As in (G), except with the addition of hnRNP B1. (K) Quantification of (J) (n = 3). (C, E, F, H, I, and K) Percent relief of inhibition is normalized to H3 signal and relative to no RNA and described as percent relief from HOTAIR-only reaction. Error bars represent SDs. P values were determined using unpaired t tests with a 95% confidence interval.

Fig. 7. Model for HOTAIR-mediated chromatin silencing via intermolecular RNA-RNA interactions.

Binding of PRC2 to single-stranded regions of HOTAIR inhibits enzymatic activity. B1 promotes RNA-RNA matching of HOTAIR with target nascent RNA via bridging of the RNAs and conformational changes in the lncRNA to promote intermolecular base pairing. B1 dissociates from the RNAs, promoting a conformation that may reduce PRC2 affinity for those RNAs through formation of the dsRNA match, thereby increasing PRC2 interaction with chromatin, leading to H3K27me3 and transcriptional repression.