Ribosomes stalling on uORF1 in the Xenopus Cx41 5' UTR inhibit downstream translation initiation

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Abstract
Translation of Xenopus laevis Connexin41 mRNA is strongly controlled by the three upstream open reading frames (uORFs) in its 5' untranslated region. Mutation of uAUG1 into AAG induced a 100-fold increase in translation of a green fluorescent protein (GFP) reporter ORF. The termination codon of uORF1 was mutated and the uORF was linked in-frame with the GFP ORF, enabling visualisation of initiation at uAUG1 by synthesis of an elongated GFP form. Unexpectedly, hardly any elongated GFP was made, suggesting that translation of uORF1 in wild-type mRNA causes constraining of the entry of 40S ribosomal subunits upstream of uORF1. A rare leucine codon, the third codon of uORF1, contributed to the slow translation and thus to slow scanning. Replacement of the rare leucine codon in uORF1 with a common leucine codon stimulated GFP translation. Remarkably, the rare leucine codon, the termination codon of uORF1, uAUG2 and uAUG3 all improved recognition of uAUG1. Apparently, the block formed by a stalled ribosome on any element in uORF1 prevented the landing of new ribosomal subunits next to the cap and therefore downregulated GFP translation.

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