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William Marshall Johnson, Inas F Aboobakar, Laura Finnegan, R Rand Allingham, Michael A Hauser, W Daniel Stamer; Identification of the hnRNPL and LOXL1-AS1 lncRNA complex: Implications for pathology in exfoliation glaucoma. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5590.
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© ARVO (1962-2015); The Authors (2016-present)
Exfoliation glaucoma (XFG) is a clinically aggressive and genetically distinct form of glaucoma caused by extrafibullary material deposition in the trabecular meshwork, leading to increased intraocular pressure and death of retinal ganglion cells. Gene variants located in the promoter of a novel long non coding RNA (lncRNA), LOXL1-AS1 associate with XFG risk. Since lncRNAs can impact susceptibility to disease through alterations in gene expression, we investigated the downstream targets of LOXL1-AS1.
RNAseq was performed on cDNA derived from an immortalized lens epithelial cell line (B-3) after RNAi knockdown of LOXL1-AS1 or scrambled control to identify differentially expressed RNAs. To identify LOXL1-AS1 binding partners, we incubated biotinylated LOXL1-AS1 or control RNA with nuclear lysates followed by a pulldown using streptavidin beads. Proteins were resolved by SDS-PAGE, and identified through analysis of mass spectrometry data. Recombinant protein candidates and LOXL1-AS1 were incubated and immunoprecipitated in vitro to test direct binding and confirm mass spectrometry results.
RNAseq identified 88 significantly altered RNAs, including 14 lncRNAs using parameters of logFC +/- 1.5 and P < 0.0001. Notably, several altered RNAs were filamentous/extracellular matrix proteins or involved in ECM homeostasis including INA, KRT14, and MMP9. To assess the mechanism by which LOXL1-AS1 alters gene expression, mass spectrometry revealed that LOXL1-AS1 interacts with the RNA processing proteins hnRNPL and ERF3A. Using protein/RNA binding studies, we demonstrated the direct binding of hnRNPL to LOXL1-AS1, through a 14 bp region in LOXL1-AS1. Conversely ERF3A, which was also identified by the mass spectrometry screen, bound to both LOXL1-AS1 and a complement RNAs indicating limited sequence specificity (false positive).
We observed that LOXL1-AS1 regulates a specific subset of target RNAs, and that this regulation may occur through interaction with hnRNPL. These findings strengthen the hypothesis that LOXL1-AS1 is a key player in XFG pathology and that targeting of the LOXL1-AS1/hnRNPL complex may provide novel therapeutic strategies for XFG.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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