Figure 5.

let-7 miRNAs suppress expression of regeneration and pluripotency-associated genes. (a) hspd1 in situ hybridization and glutamine synthetase (GS) immunofluorescence shows hspd1 RNA co-localizes (arrows) with GS-expressing MG in the uninjured retina. (b) RT-PCR shows hspd1 mRNA expression in uninjured retina that is increased following retinal injury, while pax6_b expression is undetectable in the uninjured retina and dramatically induced following retinal injury. (c) Real-time PCR quantification of hspd1, pax6_a and pax6_b mRNA levels in uninjured and 4 day post injured retina (injured). Data represent means ± s.d. (n=3 fish; compared to uninjured retina, P=0.0001 or less for hspd1, pax6_a and pax6_b, respectively, at 4 dpi. (d) let-7-dependent suppression of zebrafish proteins that are necessary for retina regeneration or associated with pluripotency. HEK 293 cells were transfected with flag or myc-tagged regeneration or pluripotency-associated gene expression vectors (50 ng) with increasing concentrations (0, 50, 200, 500 ng) ubC:let-7a,let-7f along with pCS2:mCherry for normalization. (−) lane indicates untransfected cells. Forty-eight hrs later cells were harvested and proteins resolved by denaturing SDS-PAGE. Western blots were probed with anti-mCherry, anti-Flag or anti-Myc antibodies. let-7-dependent regulation of protein expression was quantified by densitometry (Supplementary Information, Fig. S7). Experiments were repeated 3 times with similar results. See Fig. S9 for full scans. (e) Constructs used to overexpress let-7 and investigate the function of putative let-7 miRNA binding sites in the hspd1 3’ UTR. Mutations in the let-7 binding site seed sequence are indicated. (f) The hspd1 3’ UTR let-7 binding site at position 1970 confers let-7-dependent regulation on luciferase expression. Luciferase activity from 24 hpf zebrafish embryos that were co-injected at the single cell stage with the luciferase reporter and the let-7 miRNA expression vector described in (e) along with capped Renilla luciferase mRNA for normalization. Data represent means ± s.d. (n=3 fish; compared to wt 3’UTR construct in absence of pri-let-7 miRNA or mut 3’ UTR construct with pri-let-7 miRNA, P=0.0001 or less for luciferase-wt 3’ UTR activity treated with 1, 5 and 20 ng of ubC:let-7a,let- 7f construct.