Figure 4: eIF1A augments Ago2-mediated RNAi.

(a) Sequence of the human HMGA2 3′-UTR fragment mutant with two native let-7 target sites. Filled circles: Watson–Crick complementarity; open circles: non-Watson–Crick complementarity. Mutated nucleotides are shown in blue and wild-type nucleotides were underlined (to neutralize site 2 and to reduce self-complementarity). (b) In vitro effects of eIF1A on Ago2-mediated RNA cleavage. eIF1A or the indicated mutants were incubated with Ago2, synthetic let-7a and 5′-³²P end-labelled HMGA2 3′-UTR mutants. RNAs were analysed by northern blot and autoradiographs analyses. (c) Statistical analyses of three independent in vitro assays in b. (d) Western blot assays of effects of eIF1A and eIF1A (K56A) mutant on GFP-let-7 reporter expression. Ago2, eIF1A or eIF1A (K56A) were transiently overexpressed in HEK293 cells stably expressing reporter GFP-let-7. (e)The expression ratio between tubulin and GFP reporter in the same gel were calculated. Intact (100%) RISC activity was considered in control cells. The relative RISC activities under overexpression conditions of the indicated genes or mutants were obtained by comparison with the control. (f) Relative GFP reporter mRNAs in d by quantitative real-time PCR (qPCR). (g) Western blot assays of zebrafish c-Myb in the indicated zebrafish groups at 28 h.p.f. (control, eif1axb MO (62.5 μM, 1 nl), eif1axb MO (62.5 μM, 1 nl) plus human eIF1A (heIF1A) (mRNA, 1 μg μl⁻¹, 1,000 pl) and eif1axb MO (62.5 μM, 1 nl) plus heIF1A (K56A) mutant (mRNA, 1 μg μl⁻¹, 1,000 pl)). Proteins were extracted from >10 zebrafish per group. (h) Relative c-Myb mRNAs in each group in g by qPCR. Total RNAs were extracted from >10 zebrafish per group. (i) Taqman PCR assays of relative zebrafish miR-126 (dre-miR-126) in each group in g. Total RNAs were extracted from >10 zebrafish per group. In all statistical comparisons, three independent experiments were performed (mean±s.d., n=3 experiments, Student’s t-test). ***P<0.01.