Figure 2: eIF1A binds to the MID domain of Ago2 in a RNA-binding-independent manner.
From: eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference
(a) Human eIF1A interacts with the MID fragment of human Ago2. Recombinantly expressed and Benzonase-treated Ago2 fragments and recombinantly expressed GST-eIF1A were used for GST pull-down analyses followed by western blotting with anti-His antibodies (IB, immunoblotting). The arrow shows that the MID fragment interacts with eIF1A. (b) The globular domain (GD) of eIF1A interacts with Ago2. Recombinantly expressed and Benzonase-treated GST, GST-eIF1A and mutants (ND, 25∼144 aa; CD, 1∼114 aa; GD, 25∼114 aa) were used for GST pull-down assays with cell lysates of HEK293 stably expressing Ago2. (c) 1H–15N transverse relaxation optimized spectroscopy (TROSY)–heteronuclear single-quantum coherence (HSQC) spectrum of 15N-eIF1A titrated with the Sumo-MID (432∼575 aa). The titration of RNA-free-Sumo-MID leads to broadening of RNA-free 15N-eIF1A resonances (molar ratio of 1:1). Inserted frames show the broadening of resonances of V55, K56 and K67 residues in eIF1A upon MID titration. (d) 1H–15N TROSY–HSQC spectrum of 15N-Sumo-MID titrated with eIF1A. The titration of eIF1A (molar ratio of 1:1) results in broadening 1H–15N TROSY–HSQC crosspeaks coming from the 15N-MID domain, but not from 15N-Sumo (SMT3) tag. Inserted frames show the broadening of resonances of residues from the MID domain, but not from SMT3-Tag (SUMO-tag), upon eIF1A titration.
