Figure 6.

eIF3d binding to the 5′ UTR of msl-2 correlates with cap-independent repression. (A) Cap-dependency of repression of full length (mLm) and minimal (BLEF) msl-2 reporters. BLEF contains nucleotides 270–339 and 909–954 of the msl-2 5′ and 3′ UTRs, respectively (see also Figure 1A). Left, in vitro translation reactions were performed as described in the legend of Figure 1B using A-capped or m⁷G-capped mRNAs. The distance between the A and m⁷G repression lines (ΔA-G) was defined as the cap-dependency of the construct. Error bars represent the standard error of three independent experiments. Basal translation among m⁷G-capped (middle) or A-capped (right) construct was comparable. Notice the difference in absolute translation levels (ordinates) of m⁷G-capped versus A-capped constructs. (B) Analysis of cap-dependency of repression of msl-2 derivatives. Only the data obtained at SXL/RNA ratio = 40 are shown for simplicity. Error bars represent the standard error of at least three independent replicates. (C) eIF3d binds to msl-2 5′ UTR. Left, radiolabeled 5′ UTRs of constructs 5, 6 and 10 were incubated with Drosophila embryo extract in the presence (+) or absence (–) of HA-eIF3d and pulled-down with αHA beads. Immunoprecipitated material was resolved by SDS-PAGE. Right, quantification of four independent experiments. Error bars represent the standard deviation. (D) Effect of region 5 mutation on translational repression of constructs containing long versus short 5′ UTRs. Where it applies in this figure, significance was determined by unpaired Student's t-test (*P< 0.05, **P < 0.01, ***P < 0.001).