MiR-182 and miR-96 co-regulate Rac1 expression in the mouse retina

Abstract

Purpose: Previous work highlighted changes in levels of miR-1, miR-133, miR-142, miR-96, miR-182 and miR-183 in the retina of RHO-347+/-Rho+/- (R347) mouse, i.e. a rodent model for rhodopsin-linked retinitis pigmentosa. In this study, we aimed to identify proteins, whose expression is regulated by the above miRs. The strategy was to compare the proteome of the R347 and wild type (wt) retinas, and relate this to altered expression of predicted targets for the above miRs.

Methods: In silico analysis was used to predict miR targets. Protein levels from retinas of 1-month old R347 and wt mice were analysed by label free LC-MS/MS. In vitro 3’-UTR assay was used to analyse interaction between target mRNA and miR. MiR capture was used to demonstrate direct mRNA-miR interactions in vivo.

Results: 1146 unique mRNAs were predicted as targets for the above miRs. Total and membrane enriched protein fractions were prepared from pooled R347 and wt retinas. LC-MS/MS identified 1903 proteins in the retinal samples. From the predicted targets 145 were detected by LC-MS/MS; 47 of these exhibited altered expression (p<0.05) between R347 and wt retinas and were manually curated against the targeting miRs. A notable candidate from these studies was Rac1, which was selected for further exploration. Rac1 protein level increased to 339.9±24% (p<0.001) in R347 vs. wt retinas, while Rac1 mRNA expression did not change. Note that the Rac1 3’-UTR contains a highly conserved overlapping binding site for miR-96/miR-182 and the level of these miRs decrease by approx. 50% in the R347 vs. wt retina. A 3’-UTR assay on the Rac1 3’-UTR demonstrated approx. 50% suppression (p<0.001) by both miR-96 and miR-182 in vitro. MiR capture was performed on fixed wt retinas; Rac1 mRNA was enriched by 50.0 ±9.4 fold (p<0.01), while miR-96 and miR-182 levels demonstrated a combined increase of approx. 8.7-fold (p<0.05), providing evidence that these miRs are in direct physical contact with the Rac1 mRNA.

Conclusions: Results indicate that Rac1 expression is co-regulated by miR-96 and miR-182 in the mouse retina. MiR-96 and miR-182 are abundant in the retina, yet their role is not fully understood. Rac1 is also an important, yet not fully characterized, component in both photoreceptor and non-photoreceptor cells in the retina. The work provides evidence of a link between miRs dysregulated in the R347 retina and Rac1, which may play a role in certain forms of retinal degenerations.