2-D Structure of the A Region of Xist RNA and Its Implication for PRC2 Association
(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.