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. 2015 Mar 15;29(6):630–645. doi: 10.1101/gad.257048.114

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© 2015 Hu et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 5. — p54^nrb methylation by CARM1 alters its binding to dsRNAs. (A) CARM1 knockdown affects p54^nrb binding to dsRNAs but has little effect on ssRNAs. Biotin-labeled dsRNAs (double-stranded-egfp 76–495) or ssRNAs (single-stranded-egfp 1–798) were made from in vitro transcription (IVT). Nuclear extracts of scramble or CARM1 knockdown (KD) HeLa cells were incubated with biotin-dsRNA or biotin-ssRNA followed by anti-biotin pull-down and Western blotting with antibodies for anti-p54^nrb and anti-PSF. (Right) The quantitative analyses of the left panels from three independent experiments. (B) Methylated p54^nrb binds to few dsRNAs. (Left) The 620me antibody was developed to specifically recognize the methylated p54^nrb. Western blotting analyzed the specificity of 620me antibody purified from rabbit serum immunized with methylated peptides of R357 and R365. p54^nrb and Actin antibodies were used as controls. (Right) The methylated p54^nrb (620me) was enriched by biotin-ssRNA but not by biotin-dsRNA pull-downs from wild-type HeLa cell nuclear extracts. See A for details. (C) Lysine replacement of Arg357, Arg365, and Arg378 (R357K, 365K, and R378K) of p54^nrb (p54^nrb-MUT) increases the association of p54^nrb and dsRNAs. (Right) The quantitative analyses of the left panels from three independent experiments. See A for details. (D) A schematic drawing of different truncations of p54^nrb used in E and F. (E) A different domain of p54^nrb selectively binds to ssRNAs or dsRNAs. (Top) Nuclear extracts of HeLa cells transfected with different p54^nrb truncations were incubated with biotin-dsRNAs or biotin-ssRNAs followed by anti-biotin pull-down and Western blotting with anti-Flag. (Bottom) The quantitative analyses of the top panels from three independent experiments. (F) Partially purified truncation of p54^nrb selectively binds to IRAlus RNAs. Different p54^nrb truncations shown in D were purified and incubated with Dig-labeled IRAlus RNAs. See Figure 4E for details. In A, C, and E, error bars represent ±SD in triplicate experiments. P-values from one-tailed t-test in the pairwise comparison are shown.