Figure 3

The avian MYB 3'UTR contains a functional miR-150 target site. (A) Predicted binding structures between gga-miR-150 and the four putative target sites (s1, s2, s3, s4) or their mutated counterparts (mt1, mt2, mt3, mt4) are shown. Numbers correspond to the position of the site in the avian MYB 3'UTR (nt1 = first nt downstream from the stop codon); nt in bold are involved in nt pairing. (B) gga-miR-150 repressed MYB reporters in chicken DF-1 cells or human 293FT cells through one specific target site. DF-1 or 293FT cells were cotransfected with 100 ng of reporter, 300 ng of effector and 1.25 ng of normalising pcDNAMLuc plasmid. Luciferase assays were performed as in Fig. 2 and luciferase activity following transfection with pmiR-150mut was set at 100%. Reporter plasmids: MYB WT reporter, MYB mt1, mt2, mt3, mt4, mt1234 reporters. Effector plasmids: pmiR-150, pmiR-150mut. The data presented are means ± SDs of three independent experiments (* significant repression, P < 0.05, Student's t-test comparisons with pmiR-150mut controls).