Survivin (BIRC5) is an anti-apoptotic protein that is important in cancer. Mechanisms responsible for controlling Survivin levels in cells include transcriptional regulation and modulation of protein stability via post-translational modifications; however to date, translational control has been poorly studied. Here, we focused particularly on the primary control elements present in the Survivin 5' untranslated region (5'UTR). Bioinformatic analysis of ribosome occupancy on the Survivin 5'UTR revealed the presence of elongating ribosomes upstream of the canonical initiator AUG, suggesting an alternative upstream initiator AUG (uAUG) might exist. This uAUG was found out-of-frame at position -71 and appeared as a conserved element in mammals. RACE analysis revealed different transcriptional start sites for BIRC5, which indicated that translational control by this uAUG is restricted to longer 5'UTR variants. We studied the activity of the uAUG in different cell types by cloning the Survivin 5'UTR DNA sequence (wild-type and mutated variants) upstream of renilla luciferase (RLuc) into a pcDNA3 plasmid. Changes in RLuc activity were determined by luminescence assays and Western blotting. Results showed that when this uAUG was mutated to AUU or AGG in the cloned Survivin 5'UTR, RLuc activity was significantly increased. Similar results were obtained when uAUG was positioned inframe with the RLuc initiator AUG. Immunodetection of Renilla (35 kDa) by Western blotting revealed the presence of a second band (37 kDa approximately) in cells transfected with the Inframe reporter constructs, indicating that the uAUG was functional in our experimental conditions. In conclusion, our experimental data demonstrate the presence of an alternative and inhibitory initiator uAUG in the Survivin 5' UTR. This inhibitory uAUG may help understanding how Survivin expression is downregulated under physiological or pathological conditions. (C) 2020 Elsevier Inc. All rights reserved.