Abstract
Purpose :
Long non-coding RNAs (lncRNAs) are emerging as important regulators in cells and tissues. Their mechanisms of action involve interactions with genomic DNA, RNA, and proteins. We showed that ARPE-19 cells cultured for 4 months exhibited native RPE phenotype along with expression of genes, proteins, and miRNAs preferentially expressed in RPE. Furthermore, RT-PCR showed a >200-fold increase in expression of the lncRNA LINC00276 in differentiated cells. As interactions of lncRNAs with RNA-binding proteins play an important role in regulating gene expression and other cellular functions, we wished to identify interacting protein partners of LINC00276 involved in mediating RPE differentiation.
Methods :
ARPE-19 cells grown in MEM alpha medium with 1% FBS were differentiated for 4 weeks. Cell lysates from these cells were used for RNA-pulldown/mass spectrometry. Sense and antisense RNAs were synthesized by in vitro transcription of linearized LINC00276 plasmid using T7 or T3-specific RNA polymerases. Biotinylated RNAs were incubated with differentiated ARPE-19 lysates, and protein complexes were pulled down with streptavidin-coated magnetic beads and analyzed by Western blotting and mass spectrometry. RNA immunoprecipitation coupled to RT-PCR was used to further substantiate LINC00276 binding to proteins.
Results :
Pull down of proteins from differentiated ARPE-19 lysates by biotinylated RNAs followed by mass spectrometry identified PURB, SART3, ANXA6, and hnRNPL as potential LINC00276-interacting protein partners. Following further immunoblot analyses, only hnRNPL, one of the heterogeneous nuclear ribonucleoproteins (hnRNPs), a large family of RNA-binding proteins, was found to be a valid LINC00276-interacting partner. Western blot analysis of differential protein binding of sense and antisense biotinylated LINC00276 probes further validated hnRNPL binding to LINC00276. Additionally, RT-PCR of RNAs immunoprecipitated with hnRNPL antibody detected enrichment of LINC00276 but not of control GAPDH mRNA.
Conclusions :
Our results demonstrate binding of LINC00276 to hnRNPL in lysates of differentiated ARPE-19 cells. Thus, the involvement of LINC00276 in RPE differentiation appears to be mediated through interaction with hnRNPL. We are currently determining the potential role played by hnRNPL in regulating LINC00276 expression and function during ARPE-19 cell differentiation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.