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Clayton Santiago, John D Ash; Expression of the neuroprotective cytokine, Leukemia Inhibitory Factor, is potentially regulated by elements in its 3’untranslated region. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3612.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptors under stress release signals that stimulate Müller glial cells to induce the expression of trophic factors such as Leukemia Inhibitory Factor (LIF). We have observed that LIF can delay the degeneration of injured photoreceptor cells by activating survival pathways. We have also shown in mice with inherited retinal degeneration mutations that LIF expression decreases over time, which accelerates photoreceptor cell death. Understanding the mechanism by which LIF expression is reduced is key to understanding factors that control the rate of retinal degeneration. The aim of this study is to identify elements in the LIF 3’ untranslated region (UTR) that are involved in the down regulation of LIF expression.
The full length mouse LIF 3'UTR as well as its sub fragments were PCR amplified and ligated into the 3’ UTR of the renilla luciferase gene in the psiCHECK plasmid. Plasmids were co-transfected along with the control pGL4.13 plasmd into rMC-1 Müller glial cells using Lipofectamine LTX. Reporter activity was measured utilizing the Dual-Luciferase Reporter Assay System on the SpectraMax M3 microplate reader. Potential miRNA binding sites within the LIF 3’UTR were identified by utilizing the miRanda algorithm found at microRNA.org. GeneArt Site-Directed mutagenesis was employed to introduce mutations or deletions within the sub fragments. Data were normalized to the signal obtained from the co-transfected control and compared to the psiCHECK values.
The 3’UTR as well as individual sub fragments have a repressive effect on reporter activity. The 3’UTR-B fragment provides suppression identical to the full length 3’UTR. Analysis of the 3’UTR-B fragment identified 12 miRNA binding sites with high predictive scores. Mutating the majority of these binding sites did not substantially decrease suppression activity.
Our data suggests that the 3’B fragment contains elements which might confer the majority of LIF suppression. Mutations of various miRNA seed sequences found within the 3’UTR-B construct suggests that miRNAs might only play a minor role in regulating LIF expression and that other mechanisms such as RNA binding proteins might be key players in this regulation.
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