Figure 3: Lys56 in eIF1A is important for full-length eIF1A–Ago2 interaction, but K56A mutation in eIF1A does not impair translation initiation.
From: eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference
(a) Immunoprecipitation (IP) assays were performed with anti-Flag antibodies in HEK293 cell lines stably expressing Flag-tagged eIF1A and eIF1A mutants. Bottom panel: immunoprecipitation assays were performed with anti-Ago2 antibodies. Immunoblotting (IB) assays were performed with anti-eIF1A and anti-Ago2 antibodies. (b) 0.3 μg added purified Flag-eIF1A (K56A) mutants per reaction display similar promotion effects as 0.3 μg added purified Flag-eIF1A and Flag-eIF1A mutants per reaction in cap-dependent and IRES-dependent translation in dual-luciferase assays in vitro. Relative luminescence results are shown. Three independent experiments were performed (mean±s.d., n=3). (c) Polysome profiling analyses with HEK293 cell lines stably expressing eIF1A or eIF1A (K56A). Extracts of the indicated cell lines were subjected to sucrose density gradient centrifugation. The ribosome profiles (recorded at 260 nm) and the positions of the 40S/60S ribosomal subunits, 80S monosome and polyribosomes are shown. WT, wild type.
