Figure 5. NcRNAs-Pc2 Interactions Underlie Relocation and Transcriptional Activation of Growth Control Genes.

(A) Methylated and unmethylated Pc2 bind to RNAs. CLIP assay using Pc2K191me² (left panel) or Pc2K191 (right panel) antibodies were performed and specific protein-RNA complexes (indicated by red box) were visualized by autoradiography. (B) Validation of interactions of TUG1 and NEAT2 with Pc2. In vitro transcribed biotinylated TUG1 RNA (left panel) or NEAT2 RNA (right panel) were incubated with HeLa nuclear extract. The elution of Monoavadin beads was subjected to immunoblotting with indicated antibodies. (C) The selective binding of Pc2 to TUG1 or NEAT2 depends on K191 methylation state. RNA gel shift assay was performed using synthesized TUG1, NEAT2 RNA and their corresponding DNA oligos and recombinant His-Pc2 with or without in vitro methylation. (D–F) TUG1 and NEAT2 function as modulators of Pc2 chromodomain for “reading” the histone code. MODified Histone Peptide ArrayR was incubated with Recombinant His-Pc2 chromodomain in the presence of yeast tRNA (D), in vitro transcribed TUG1 (117-3390) (E), or NEAT2 (2281-5611) (F) RNA fragments and subjected to immunoblotting with indicated antibodies. The Myc tag antibody was added to detect Myc tagged peptides on position P21 as positive control. The binding specificity calculated by Array Analyses Software based on two arrays (images shown in Figures S5F–S5H) was shown. (G) TUG1 is localized in PcG bodies. HeLa cells microinjected with molecular beacon probes targeting TUG1 were subjected to immunostaining with Pc2K191me² antibodies. (H) TUG1 and NEAT2 are required for serum-induced relocation of the MCM3 locus. HeLa cells microinjected with validated siRNA targeting TUG1 or NEAT2 were serum-starved followed by restimulation and Immuno-FISH analyses were performed using Ring1A, SC35 antibodies and probes targeting MCM3 locus. Statistical analyses on colocalization of MCM3 loci with Ring1A staining (left panel) and SC35 staining (right panel) are shown. (I and J) Effect of TUG1 or NEAT2 knockdown on serum-induced growth control gene expression and cell proliferation. HeLa cells transfected with TUG1 or NEAT2 siRNA were serum-starved followed by restimulation and relative mRNA levels of indicated genes and cell proliferation were determined by qRT-PCR (I) and flow cytometry (J). (K) A list of TUG1 and NEAT2 ncRNA- associated proteins identified from two independent MS experiments. Mean±SEM, *p<0.05, **p<0.01 and ***p<0.001.

See also Figure S5 and Table S1–S3.