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D. O'Brien, H. Huang, V. Canto-Soler; Analysis of Post-Transcriptional Regulation of Muscleblind-Like 1 Expression in Retinal Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5936.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies suggest that the expression of the zinc finger-containing protein Muscleblind-like 1 (MBNL1) may be regulated post-transcriptionally in the developing retina (Huang et al. Dev Dyn, 2008). We have hypothesized that the 3’ untranslated region (3’UTR) of MBNL1 plays a key role in this phenomenon and is a target for trans-regulatory elements. The purpose of this study is to investigate these possibilities.
Highly conserved regions of the 3’UTR of MBNL1 were identified by Blast analysis. The complete MBNL1 3’UTR, or segments containing the conserved regions, were cloned into the pcDNA3-mRFP expression vector. In these constructs, monomeric red fluorescent protein (mRFP) is driven by a CMV promoter and the 3’UTRs are cloned into the 3’-end of the mRFP open reading frame. A similar plasmid containing NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 (NDUFA10) 3’UTR was used as control. COS-7, HEK293, and ED6 chicken retinal cells in culture were co-transfected with these constructs and a plasmid containing enhanced green fluorescent protein (EGFP) as a reporter control gene. EGFP and mRFP expression were analyzed at the mRNA level by semi-quantitative PCR. Protein levels were analyzed by Western blot and by the intensity of their fluorescence in live cells measured by image analysis software.
In the presence of control 3’UTR, expression of mRFP was similar to that of EGFP. However, incorporation of the complete MBNL1 3’UTR, or certain conserved regions, caused inhibition of protein synthesis without affecting mRNA levels. In retinal cultures, this inhibitory effect was more significant in photoreceptors than in non-photoreceptors, and in cells derived from the fundus than in those from the periphery. Based on the differential inhibitory effect seen between several of the 3’ UTR MBNL1 constructs, we have identified a region that seems to be responsible for most of the effect. Several miRNAs candidates, including a few targeting this region, have been also identified by bioinformatics analysis.
These results suggest that MBNL1 3’UTR is a cis-regulatory element and modulates repression of MBNL1 protein synthesis. We are now carrying out experiments to verify these mechanisms in the retina in vivo and to test candidate miRNAs that could potentially target the MBNL1 3’UTR in developing retinal cells.
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