Purchase this article with an account.
Andrei Surguchov, Irina Surgucheva; Post-transcriptional regulation of γ-synuclein expression and its role in glaucomatous alterations. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1608.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is related to the accumulation of aggregated and misfolded proteins. γ-Synuclein (γ-Syn) accumulates in the retina and in the optic nerve astrocytes in large spheroidal structures in patients with glaucoma and in animal models forming protease resistant intracellular inclusions. Defects in the regulation of γ-Syn expression are responsible for the accumulation of such pathological deposits. microRNAs (miRNAs) are small, non-coding RNAs, that regulate gene expression through their targets in the 3'untranslated regions of mRNAs. Here we investigate the mechanism of post-transcriptional regulation of γ-Syn expression by miRNAs.
A 275 bp fragment was generated by PCR using human γ-Syn clone (long form, LF). LF and modified forms of this fragment with deleted putative miRNAs targets were inserted in a Luc reporter vector and Luc activity was tested after transient transfection. Expression of miRNAs was carried out using “BLOCK-iT Pol II miR RNAi Expression Vector”. miRNAs were quantified using qRT-PCR with TaqMan probes.
First we analyzed γ-Syn 3’-UTR by bioinformatics approach and identified putative targets for the following miRNAs: miR-103, 107, 424, 497, 4437 and 4674 and 4722. The insertion of LF downstream of the LUC gene caused a 51% reduction of LUC activity, confirming the presence of effective targets for miRNAs in this fragment. Deletion of targets for miR-103 and miR-107 from LF does not change the activity, although according to qRT-PCR data these miRNAs are present in tested cell cultures. The effect of truncated form (TF) of the 3’-UTR in which we have modified targets for miR-4437 and miR-4674 on LUC activity was different: it did not change LUC activity, while the deletion of miR-103 targets from this TF increases the activity. To further investigate the effect of miR4437 and miR4674 on γ-Syn expression we performed expression of these two miRNAs in SK-BR3 cells. According to Western blotting data, expression of miR4437 caused a 69% and miR4674 a 76% reduction of endogenous γ-Syn expression. On the other hand, in stable clones overexpressing γ-Syn no significant effect of miRNAs on γ-Syn expression was found.
3’-UTR contains targets for miRNAs which are used for the regulation of γ-Syn expression. This mechanism of regulation is cell-specific and depends on the level of γ-Syn and miRNAs expression.
This PDF is available to Subscribers Only