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H. Huang, V. Canto-Soler, R. Adler; Post-Transcriptional Regulation of Muscleblind-Like 1 (mbnl1) Expression in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5880. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies suggested that the expression of the zinc finger protein MBNL1 may be regulated post-transcriptionally in the retina (Huang et al. Dev Dyn, 2007. In press). We have hypothesized that the 3’ untranslated region (3’UTR) of the MBNL1 mRNA plays a key role in this phenomenon, and is potentially targeted by microRNAs. The purpose of this study is to investigate these possibilities.
The 3’UTR of MBNL1 and of a control gene (NDUFA10) were cloned into the pcDNA3-mRFP expression vector through the Gateway strategy and confirmed by sequencing. In these constructs, the monomeric red fluorescent protein (mRFP) gene is driven by a CMV promoter, and the cloned 3’UTR is in a 3’-end position relative to the mRFP open reading frame. Plasmids were transferred into cultured cells by lipid-mediated transfection and into early chick embryo retinas (st 9-12) in ovo by electroporation. An EGFP plamsid without 3’UTR was co-transfected or co-electroprated for comparison. mRFP and EGFP expression were analyzed at the mRNA level by semi-quantitative PCR and in situ hybridization, and at the protein level by their own fluorescence and by Western blots and immunocytochemistry.
Blast results demonstrated that chicken MBNL1 3’UTR has three domains that are highly conserved in other species, suggesting their functional relevance. In the absence of a 3’UTR, or with a control 3’UTR, patterns of mRFP expression were similar to those of EGFP. However, incorporation of different regions of the MBNL1 3’UTR caused inhibition of mRFP protein synthesis, without affecting the levels of mRFP mRNA. Equivalent inhibition was observed in embryonic retina in ovo, in primary retinal cell cultures, and in COS-7 cells, but not in HEK293 cells.
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