Fig. 1

Linc-RAM directly interacts with PYGM in the cytoplasm. A-F Linc-RAM in cytoplasmic (Cyto), nuclear-soluble (Nuc.Sol), and nuclear-insoluble (Nuc.Insol) fractions of C2C12 cells cultured in growth medium (A-C) and differentiation medium for 24 h (D-F), as determined by RT–qPCR. The GAPDH mRNA was used as a marker for the cytoplasmic fraction. Neat1 (nuclear paraspeckle assembly transcript 1) was used as a marker for the nuclear fraction. The data are representative of three independent experiments. G Schematic diagram showing the strategy applied to identify Linc-RAM-binding proteins using MS2-MBP-mediated RNA pull down. Three bacteriophage MS2 coat protein-binding sites (3 × MS2 hairpins) were fused to the 3′-end of Linc-RAM (Linc-RAM-3 × MS2). MS2-MBP represents a fusion protein comprising MS2 coat protein and maltose-binding protein. H A representative silver-stained SDS-PAGE gel showing the bands that differed (red arrow) between Linc-RAM-3 × MS2 (Linc-RAM) and the 3 × MS2 control (Ctrl). The differential bands were individually extracted and subjected to mass spectrometry (MS) analysis. I, J RNA immunoprecipitation (RIP) analysis to validate the physical interaction between Linc-RAM and PYGM in C2C12 cells cultured in differentiation medium for 24 h. Native (I) or UV-crosslinked (J) C2C12 cells differentiated for 24 h were immunoprecipitated using anti-PYGM, anti-MyoD, and IgG antibodies. Linc-RAM in immunoprecipitates was examined by RT–qPCR. GAPDH served as a negative control. MyoD antibodies served as a positive control, as we previously reported that Linc-RAM binds MyoD (Yu et al., 2017). K Representative RNA electrophoretic mobility shift assay (EMSA) results obtained using biotin-labeled Linc-RAM and different doses of recombinant GST-PYGM fusion protein (50 ng, 100 ng, 200 ng). The biotin-labeled Linc-RAM and recombinant PYGM protein complex were resolved on a 5% native polyacrylamide gel and subsequently the Linc-RAM/PYGM complex was detected by HRP-Streptavidin. Recombinant GST protein (GST-only) served as a negative control. L Representative RNA EMSA results obtained using biotin-labeled Linc-RAM and recombinant GST-PYGM fusion protein (200 ng). As competitors, non-labeled Linc-RAM probes were added to confirm the binding specificity. The presented values reflect the means ± SE obtained from three independent experiments