2-D structure of the A region of Xist RNA and its implication for PRC2 association

Abstract

In placental mammals, inactivation of one of the X chromosomes in female cells ensures sex chromosome dosage compensation. The 17 kb non-coding Xist RNA is crucial to this process and accumulates on the future inactive X chromosome. The most conserved Xist RNA region, the A region, contains eight or nine repeats separated by U-rich spacers. It is implicated in the recruitment of late inactivated X genes to the silencing compartment and likely in the recruitment of complex PRC2. Little is known about the structure of the A region and more generally about Xist RNA structure. Knowledge of its structure is restricted to an NMR study of a single A repeat element. Our study is the first experimental analysis of the structure of the entire A region in solution. By the use of chemical and enzymatic probes and FRET experiments, using oligonucleotides carrying fluorescent dyes, we resolved problems linked to sequence redundancies and established a 2-D structure for the A region that contains two long stem-loop structures each including four repeats. Interactions formed between repeats and between repeats and spacers stabilize these structures. Conservation of the spacer terminal sequences allows formation of such structures in all sequenced Xist RNAs. By combination of RNP affinity chromatography, immunoprecipitation assays, mass spectrometry, and Western blot analysis, we demonstrate that the A region can associate with components of the PRC2 complex in mouse ES cell nuclear extracts. Whilst a single four-repeat motif is able to associate with components of this complex, recruitment of Suz12 is clearly more efficient when the entire A region is present. Our data with their emphasis on the importance of inter-repeat pairing change fundamentally our conception of the 2-D structure of the A region of Xist RNA and support its possible implication in recruitment of the PRC2 complex.

Figure 1. Probing of mouse Xist A region RNA structure alone and inside 5′-terminal region.

The two RNAs (A. for 5′ terminal 1137-nt long RNA, B. for A region) were in vitro transcribed and renatured as described in Material and Methods, before being subjected to limited digestion with T1, T2, or V1 RNases under the conditions described in Materials and Methods. Extension analyses were performed using oligonucleotide 3866 (Table S1) as the primer. The resulting cDNAs were fractionated by electrophoresis on 7% denaturing polyacrylamide gel. Lanes U, G, C, and A correspond to the sequencing ladder obtained with the same primer. Lanes marked by Contr corresponds to primer extension analysis of undigested RNA transcripts. Nucleotide numbering on the left-hand side of the autoradiogram takes the first residue of mouse Xist RNA as residue 1. The sequences corresponding to repeats 3 and 4 are indicated by vertical bars on the right-hand side of the autoradiograms.

Figure 2. Representation of experimental data on the previously proposed 2-D structure of the Xist A region.

Each of the seven repeats as well as the eighth half repeat from the mouse A region was folded according to the previously predicted two stem-loop structure . T1, T2, and V1 RNase cleavages were represented by arrows surmounted by circles, triangles, and squares, respectively. Nucleotides modified by DMS or CMCT are circled. Colours of circles and arrows indicate the yields of modifications and cleavages—red, yellow, and green for strong, medium, and low modification or cleavage, respectively. The V1 RNase cleavages and chemical modifications that cannot be explained by the two stem-loop structure models are encircled by blue lanes.

Figure 3. Representation of experimental data on the possible 2-D structure 1 of the Xist A region.

In model 1, the various stem-loop structures all involve two successive repeats. The repeats are indicated by red lines and are numbered from 1 to 8. Representations of chemical and enzymatic data are as in Figure 2. In each of the three panels, segments in which DMS and/or CMCT modifications were identified are indicated on a schematic drawing of the 2-D structure (full and dot lines for DMS and CMCT, respectively). In segments analyzed by the two chemical reagents, unmodified nucleotides are squared. However, one should take into consideration the fact that G residues are poorly modified by CMCT in the mild conditions that we used. The free energies of each stem-loop structures at 0°C and in 3 M NaCl were calculated with the M-fold software.

Figure 4. Representation of experimental data on the possible 2-D structure 2 of the Xist A region.

In model 2, repeats in the 5′ half of the A region interact with repeats in the 3′ half of this region. Representation of enzymatic cleavages and chemical modifications are as in Figures 2 and 3.

Figure 5. Representation of experimental data on the possible 2-D structure 3 of the Xist A region.

In model 3, two stem-loop structures containing four repeats are separated by a small stem-loop corresponding to poly A and poly U sequences. Representation of enzymatic cleavages and chemical modifications are as in Figures 2 and 3.

Figure 6. Probing of the 2-D structures of the entire and 5′ half of human A region.

Legend as in Figure 1, except that the transcripts correspond to the entire human A repeat region (human A RNA) and the 5′ half of the human A region (SLS1H RNA), respectively.

Figure 7. Representation of experimental data on possible structure 3 of human Xist A region.

Repeat 5 is located in a short central stem-loop structure flanked by two larger stem-loop structures that each contain four repeats. Representation of the enzymatic cleavages and chemical modifications are as in Figures 2 and 3. Indication of segments in which DMS and/or CMCT modifications were identified is represented as in Figure 3. The free energies of the stem-loop structures were calculated with the M-fold software.

Figure 8. Steady-state fluorescence studies provide additional support to the A region Model 3.

In (A, B, and C), the binding sites of oligonucleotides P1 to P6 used in the FRET experiments are shown for the three 2-D structures of the A region corresponding to Models 1, 2, and 3. The identity of the chromophore present in each oligonucleotide (donor Cy3 or acceptor Cy5) is indicated in green and blue, respectively. Cy3- and Cy5-labeled oligonucleotides were purchased from Eurogentec. As illustrated in (D), the emission fluorescence spectra from 530 to 745 nm of the donor oligonucleotide bound alone to the RNA (green curve) were collected, as well as the emission spectra obtained in the presence of the donor and acceptor oligonucleotides (violin curve). No energy transfer between Cy3- and Cy5-labeled oligonucleotides was detected in solution. The FRET efficiency for each pair of oligonucleotides was defined as the decrease in fluorescence of the donor at 564 nm in the presence of the acceptor. Two representative examples of FRET assays (oligonucleotide pairs P2/P4 and P3/P6) are shown in (D). The FRET efficiencies measured for the six pairs of oligonucleotides are provided in (E) (mean values of three independent experiments). Standard deviations (σ) are shown. The relative efficiencies of the FRET obtained for each oligonucleotide pairs are schematically represented in (A, B, and C) by lines joining the oligonucleotides. The thickness of the lines reflects the efficiency of the FRET effect.

Figure 9. The PRC2 complex assembles on fragments of the A region containing at least four repeats.

(A) Representation of the fusion RNAs used for formation of RNP complexes with ES cell nuclear extracts. Retention of RNA containing three MS2 coat protein binding sites (MS2) on amylose beads was mediated by the MS2/MBP fusion protein . Analysis of the protein content of the RNP complexes formed on the A region/MS2 RNA was achieved by mass spectrometry (Figure S8). (B and C) Western blot assays using antibodies specific for the PTB, Suz12, RbAp46, RbAp48, Eed, and Ezh2 protein were used to evaluate the relative amounts of these proteins in the purified complexes formed with the various RNAs shown in (A). Antibodies were purchased from Santa Cruz (Sc), Abcam (ab), and Calbiochem (cb): anti-Ezh2 (anti-ENX-1 H-80, sc-25383), anti-RbAp46 (ab3535), anti-RbAp48 (ab488), anti-Eed (ab4469), anti-Suz12 (ab12073), and anti-PTB (cb NA63). (D and E) Test of the association of radiolabelled fragments of the A region with components of the PRC2 complex present in nuclear extracts of ES cells. The three RNAs tested are represented in (D). RNAs bound to the G sepharose beads were fractionated by electrophoresis on 7% denaturing gels. Autoradiograms of the gels are shown in (E). Input corresponds to 10% of the material incubated with the beads.