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Arpad Palfi, Karsten Hokamp, Stefanie M Hauck, Sebastian Vencken, Matthew Carrigan, Marius Ueffing, Catherine Greene, G Jane Farrar; miRNA regulation and potential interactors of Rac1 in the mouse retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4801.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous work demonstrated Rac1 up-regulation in the RHO-P347S (R347) mouse retina and miR-96/182 co-targeting of Rac1 mRNA. Here we evaluated what additional miRNAs target Rac1 in the mouse retina using miRNA capture affinity technology (miR-CATCH). Moreover, to identify possible Rac1 regulated circuits in the R347 retina, we analysed expression of Rac1 interacting proteins using high-throughput proteomics.
In vivo miR-CATCH was performed in wild type (wt) mouse retina extracts and miRNA expression determined utilizing Exiqon rodent miRNA PCR panel profiling. miRNA targets were predicted employing miRSystem and RNA22. Proteome analysis from R347 and wt mouse retinas was carried out using label-free liquid chromatography-mass spectrometry (LC-MS/MS). The Rac1 interactome was constructed in InnateDB.
miR-CATCH enriched Rac1 mRNA 50.0±9.35-fold (p<0.01; n=3). miRNAs both detected to be enriched (via miR-CATCH) and predicted to target (via in silico analysis) Rac1 mRNA (3’UTR and coding region) included miR-103-3p (9.5-fold enrichment, p=0.0012), miR-9-5p (5.4-fold enrichment, p=0.038), let-7c-5p (4.3-fold enrichment, p=0.046,) and miR-183-5p (2.5-fold enrichment, p=0.033). miRSystem and RNA22 did not predict approximately half of the Rac1 miR-CATCH enriched miRNAs to target Rac1; these miRNAs included miR-125b-5p (5.2-fold enrichment, p=0.08), miR-181b-5p (3.8-fold enrichment, p=0.02) and miR-181a-5p (3.0-fold enrichment, p=0.03). Additionally, LC-MS/MS analysis of R347 versus wt retinas identified 30 proteins from 148 proteins present in the mouse InnateDB Rac1 interactome, and 9 proteins from 22 photoreceptor outer segment Rac1 interactors. From the mouse interactome, Rac1, Cav1 and Rap1gds1 were significantly up-regulated, and Nckap1 down-regulated, while from the outer segment interactors, expression of Eno1 and Sag decreased significantly in the R347 retina.
Our data define the Rac1-miRNA interactions that occur in the mouse retina and suggest significant utilisation of both canonical and non-canonical (the latter not predicted in silico) miRNA targeting of Rac1. Analysis of the Rac1 interactome in this study indicates parallel up-regulation of Rac1 and Rap1gds1 (an activator of Rac1), while Cav1 up-regulation may suggest activation of a glial Rac1 axis and possibly activation of integrin-mediated effector mechanisms in R347 retina.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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