Requirement for eIF3h for efficient reinitiation. (A) To delineate leaky scanning across uORFs 2 and 3, all in-frame stop codons were eliminated by point mutations, thus causing them to overlap the main FLUC ORF by 44 and 45 amino acids, respectively (2a,2b,3 ovlp). Residual expression of FLUC is attributed to ribosomes that have leaky-scanned uORFs 2a, 2b, and 3. (B) The effect of eIF3h on reinitiation downstream from uORF 2a,2b,3 was tested by shortening the intercistronic spacer length in the AtbZip11 leader by deletion from the 3′ end of the spacer. uORFs 2a, 2b, and 3 were placed in a strong Kozak context (−3aaaAUGg+4) to minimize the effect of leaky scanning. Error bars, standard error (n = 5–8). (*) Statistical significance at P < 0.002. (C) Inhibition of translation by an ATF4-like (Harding et al. 2000) overlap-uORF downstream from uORF2 demonstrates consistent reinitiation in wild type but marginal reinitiation in eif3h. A uORF (gray box) in a strong Kozak context (A−3AAAUGG+4) was created starting 60 nt upstream of the FLUC ORF to overlap the main ORF. (*) AUGs in a strong context. Bars, standard error (n = 8–12). AtbZip-FLUC and endogenous EF1α mRNA levels were estimated by RT-PCR.
