hnRNP A2/B1 prevents IFN-γ-induced hnRNP L prolyl hydroxylation, blocks interaction with VHL, and stabilizes hnRNP L.

<p>(A) Rapid degradation of hnRNP L in absence of hnRNP A2/B1. U937 cells were transfected with hnRNP A2/B1-specific (or scrambled) siRNA. After recovery, cells were treated with IFN-γ and Hpx. for 0, 8, and 24 h. Lysates were immunoblotted with anti-hnRNP A2/B1, -hnRNP L, and -GAPDH antibodies. (B) Time course of hnRNP L degradation in absence of hnRNP A2/B1. U937 cells were treated as in (A) for up to 24 h. Lysates were immunoblotted with anti-hnRNP L and -DRBP76 antibodies. (C) IFN-γ induces prolyl hydroxylation of hnRNP L. U937 cells were treated with IFN-γ and MG132 for up to 24 h. Lysates were immunoprecipitated with anti-hnRNP L antibody and immunoblotted with anti-hydroxyproline and -hnRNP L antibodies. (D) hnRNP L is not subject to prolyl hydroxylation in Hpx. U937 cells were treated with IFN-γ and Hpx. for up to 24 h. Lysates were immunoprecipitated with anti-hnRNP L antibody and immunoblotted with anti-hydroxyproline and -hnRNP L antibodies. (E) hnRNP A2/B1 inhibits prolyl hydroxylation of hnRNP L in hypoxia. U937 cells were transfected with hnRNP A2/B1-specific (or scrambled) siRNA. After recovery, cells were treated with IFN-γ, Hpx., and MG132 for up to 24 h. Lysates were immunoblotted with anti-hnRNP A2/B1 antibody. Lysates were also immunoprecipitated with anti-hnRNP L antibody and immunoblotted with anti-hydroxyproline and -hnRNP L antibodies. (F) Hypoxia induces binding of hnRNP A2/B1 to hnRNP L and prevents VHL binding. U937 cells were subjected to Nmx. or Hpx. for 24 h in the presence of IFN-γ stimulus. Lysates were immunoprecipitated with anti-hnRNP L antibody and immunoblotted with anti-hnRNP A2/B1 and -VHL antibodies. Total hnRNP L in cell lysates was determined by immunoblot. IP with pre-immune IgG of hypoxic lysate served as a control. (G) Reconstitution of RNA switch function of HILDA complex. Phospho-mimetic hnRNP L (Y359D) was pre-incubated with DRBP76 and hnRNP A2/B1 as indicated (5 pmol each) for 0.5 h on ice. <i>In vitro</i> translation of the FLuc reporter bearing the <i>VEGFA</i> HSR element (and <i>RLuc</i> control RNA) was determined in a wheat germ extract in the presence of ³⁵S-Met, cytosolic extracts from IFN-γ-treated U937 cells, and HILDA components as indicated. In a control experiment, wild-type hnRNP L replaced phospho-mimetic hnRNP L. FLuc expression was normalized by RLuc and reported as mean ± SD, <i>n</i> = 3.