Figure 6.
Methylation of hnRNP A1 by PRMT5 facilitates recognition of IRES-containing RNAs. PRMT5flox/flox MEFs were treated with 2 μM tamoxifen (TAM) for 8 days. (A) The cell lysates from the treated and untreated cells were incubated overnight with biotin-labeled CCND1 or MYC IRES RNA or their control antisense RNAs. RNA complexes were pulled down by streptavidin-conjugated beads and subjected to Western blotting with anti-hnRNP A1 or anti-PRMT5 antibodies. (B) The relative IRES-bound hnRNP A1 signal intensity from Figure 6A was calculated by densitometry. Signal intensity of hnRNP A1 from lane 1 was set as 1. (C) Cell lysates from tamoxifen treated or untreated cells were incubated with normal mouse IgG or anti-hnRNP A1 antibody in the presence of RNase inhibitors overnight, and immunoprecipitated with protein A/G conjugated beads. Bound RNAs were then eluted, purified and subjected to RT-qPCR for CCND1 and MYC mRNAs. (D) Wild type or double-site mutant of GFP-hnRNP A1 constructs were transfected into hnRNP A1 knockout HeLa cells. The levels of CCND1 and MYC mRNAs bound to wild type or mutated hnRNP A1 were detected by RT-qPCR, following RNA immunoprecipitation with anti-GFP antibody.