Abstract
Purpose :
Long non-coding RNAs (lncRNAs) have emerged as essential regulators of diverse cellular processes. However, the specific roles of lncRNAs in human RPE differentiation are still largely unknown. Earlier, we observed a >200-fold higher expression of lncRNA LINC00276 in differentiated ARPE-19 cells along with the expression of genes, proteins, and miRNAs preferentially expressed in RPE. As interactions of lncRNAs with RNA-binding proteins play an important role in regulating gene expression and cell signaling, we wished to identify cell signaling modulators interacting with RNA-binding proteins and LINC00276 that mediate differentiation in RPE cells.
Methods :
ARPE-19 cells grown in MEM alpha medium with 1% FBS were differentiated for 4 months. In vitro transcription was used for synthesizing sense and antisense RNAs using T7 or T3-specific RNA polymerases from linearized LINC00276 plasmid. Biotinylated RNAs were used to pulldown protein complexes from differentiated ARPE-19 lysates. Mass spectrometry (MS) and western blotting were used to analyze proteins. Human signal transduction pathway arrays followed by pulldown and RIP assays coupled with RT-PCR were used to study LINC00276 involvement in regulating the epithelial differentiation of ARPE-19 cells.
Results :
RNA pull-down followed by MS analysis identified PURB, SART3, ANXA6, and hnRNPL as potential LINC00276-interacting protein partners in the differentiated ARPE-19 cell lysates. Subsequently, immunoblot analyses coupled with MS identified hnRNPL as a valid LINC00276-interacting partner. Additionally, RT-PCR of RNAs immunoprecipitated with hnRNPL antibody detected enrichment of LINC00276 but not of control GAPDH mRNA. RT-PCR analyses of the Wnt-signal transduction pathway array along with RIP assay identified hnRNPL interacting Wnt-responsive transcription factors in the differentiated ARPE-19 cells.
Conclusions :
Our results demonstrate the binding of LINC00276 to hnRNPL in lysates of differentiated ARPE-19 cells. Furthermore, the involvement of LINC00276 in RPE differentiation appears to be mediated through a signal transduction pathway involving Wnt-responsive transcription factors. We are currently determining the potential role played by Wnt signaling molecules in regulating LINC00276 expression and function during ARPE-19 cell differentiation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.