Fig. 5

Fig. 5 Fig. 5: Identification and functional analysis of the RBP interactome for two coPARSE-lncRNAs., PCA of MS data for HeLa cell lysates pulled down for the indicated human coPARSE-lncRNAs and the predicted mouse and zebrafish homologs. The control samples are based on luciferase transcript segments. b, Distribution of the MiST scores of enriched RBPs upon pull-down using the human coPARSE-lncRNA RP1-212P9.3 and its predicted mouse and zebrafish homologs. Dashed lines represent a threshold of 0.7. Three commonly enriched RBPs from all comparisons (highlighted in colored circles) were validated by immunoblotting. The r represents the Pearson correlation coefficient, two-sided Student’s t-test. c, Venn diagram showing identified binding proteins of eight lncRNAs (human coPARSE-lncRNAs THORLNC, RP1-212P9.3 and RP11-1055B8.4, and their mouse and zebrafish homologs) in the RNA pull-down experiments (top). The table presents common binding proteins of three human lncRNAs and their homologs (bottom). Each dot represents a binding protein. d,e, Time-matched images (d) and quantifications (e) of early embryogenesis showing that injection of a human homologous coPARSE-lncRNA RP1-212P9.3 fragment and an RP1-212P9.3 fragment with the intact NONO-binding sites (RP1-212P9.3 re1) rescued the developmental defect of the corresponding zebrafish lncRNA homolog knockdown embryos. The epiboly edge is marked by red dotted lines, and the embryonic shield is indicated by red arrowheads. n = 3 biologically independent experiments. The number of embryos in each injection group is detailed in Methods. Scale bars, 100 μm. Error bars, means ± s.d., two-sided Student’s t-test. f,g, High-content imaging proliferation assays of RP1-212P9.3 (f) and RP11-1055B8.4 (g) KO HeLa cells rescued with wild-type zebrafish homologs and mutants bearing mutated RBP-binding sites. A luciferase segment was used as a negative control. AAVS1/FLU, control with pcrRNA targeting AAVS1 gene and overexpression of the luciferase segment. All groups were cultured with 500 ng ml⁻¹ Dox. Error bars, means ± s.d., n = 3 biologically independent experiments. h, A simplified model for the evolution and function of coPARSE-lncRNAs. NS, not significant.

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