eIF4GI associates with Ago2.
(A) Schematic diagram of human eIF4GI. ‘4E’ means ‘the eIF4E-binding motif’. Plasmids were constructed for expression of various Flag-tagged eIF4GI fragments in human cells. (B) The N-terminal and middle domains of eIF4GI participate in the eIF4GI-Ago2 association. Plasmids expressing Flag-tagged eIF4GI variants and myc-tagged full-length Ago2 were co-transfected in 293FT cells, and their associations were examined by Flag immunoprecipitation (Flag-IP) with the Flag-resin. The levels of Ago2 and the eIF4GI mutants (left panel) and the amount of co-precipitated Ago2 (right panel) were monitored by Western blotting using the indicated antibodies. (C) Schematic diagram of the N-terminal constructs of eIF4GI for fine mapping of the region required for the association with Ago2. (D) Determination of the Ago2-associated region in eIF4GI. WCEs from 293FT cells expressing myc-tagged full-length Ago2 and N-terminal variants of eIF4GI serially deleted from the C- or N-termini were subjected to Flag-IP. The expressions of Ago2 and the eIF4GI derivatives (lower panel) and the amount of precipitated Ago2 (upper panel) were examined using the indicated antibodies. (E) RNA-independent association of Ago2 with eIF4GI (aa 42–202). WCEs from 293FT cells expressing myc-tagged Ago2 and Flag-tagged eIF4GI-NtP were treated with (lanes 2 and 4) and without (lanes 1 and 3) RNase A and subjected to Flag-IP.