Figure 7. Presence of two start codons in the Ens-1 5′UTR.

(A) The region containing the two putative start codons in the sequence encoding the whole mRNA (NM_001080873) is represented. The ATG start codon (in red) is the authentic initiation site and generates the 54 kDa protein that has been published previously (NM_001080873.1). The GTG codon (in red) is positioned 96 nucleotides. upstream of the AUG and generates a 57 kDa protein. The purine in positions -3 or +4 from the first base of each start codon is squared (Kozak consensus sequence). (B) Putative sequence of both proteins that only differ by the 3 kDa peptide in N-terminal position represented in bold. (C) Schematic representation of the constructs used to validate the translation initiation from the GTG and the ATG start codons in the 5′ UTR of Ens-1. (D) RNA generated from the constructs depicted in (C) were used for in vitro translation of the luciferase protein in the presence of radioactive methionine. [³⁵S]methionine-labelled proteins were separated on SDS-PAGE and revealed by autoradiography. The position of the luciferase protein (LUC) generated by constructs with only one start codon is indicated (GTG in Ens-1 5′UTR or ATG in CTR). Initiation from the two start codons generates LUC with ATG and a larger protein (LUC+3 kDa) resulting from initiation at the upstream GTG.