Figure 3: Characterization of uORFs as potent cis-acting repressors in the translational regulation of signalling pathway genes.

(a) Multiple core components of Shh, Wnt and Hippo pathways that are under significant translational regulation are shown in blue. Pointed and blunt arrows indicate stimulation or inhibition. (b) Firefly luciferase (Fluc) reporter activity downstream of 5′-UTRs of Hippo or Shh pathway genes. Values are shown relative to HBB 5′-UTR construct indicated by the dashed line. Full-length wildtype 5′-UTRs contain multiple uAUGs (red arrows) and non-canonical initiation sites within a strong Kozak context (blue arrows). Black bars indicate activity of wildtype 5′-UTR, grey bars indicate activity of mutated 5′-UTRs in which point mutations were generated in all uAUGs (red triangles) and non-canonical initiation sites (blue triangles). (c–f) Values are shown relative to wildtype 5′-UTR constructs: uORFs initiating at uAUG (red arrows); uORFs initiating from non-canonical start sites (blue arrows); point mutations in uAUGs (red triangle); point mutations in non-canonical start sites (blue triangles). (c) Fluc activity downstream of wildtype and mutant Gli1 5′-UTR (d) or Ptch1 5′-UTR. (e) uORF overlapping with main ORF (oORF) is indicated by yellow arrows. Point mutation in the 4th uAUG reveals that it is not important for translational repression of Ptch1 5′-UTR. (f) uORFs that are in frame with main ORF are indicated by blue arrows; oORF (yellow arrows). A green triangle indicates the location of a point mutation (tg‘T’GA to tg‘A’GA). This point mutation disrupts the upstream stop codon for the 3rd and 5th uAUGs changing the two corresponding uORFs to oORFs and producing a new stop codon following the 4th uAUG changing its corresponding sequence from an oORF to an uORF. The insertion of 1 nt (‘Stop+1 frameshift’) just before the CDS shifts the 3rd and 5th uAUGs in frame with the main ORF. The insertion of 2 nt (‘Stop+2 frameshift’) just before the CDS shifts these uAUGs back out of frame. For data in panels (b–f) assays were performed in NIH3T3 cells. Fluc reporter activity was normalized to Fluc mRNA and transfection efficiency using co-transfected Renilla luciferase (Rluc) normalized to Rluc mRNA. Error bars represent s.d. AU, arbitrary unit; **P<0.01; *P<0.05; NS, not significant (t-test, n≥4).