The long non-coding RNA LINC00276-202 regulates RPE differentiation through hnRNPL and β-catenin-associated signaling

Abstract Non-coding RNAs play critical roles in regulating gene expression and diverse biological processes. While the roles of microRNAs (miRNAs/miRs) are well known, the roles of long non-coding RNAs (lncRNAs; >200 nucleotides, with little or no protein-coding potential) remain less defined. In this study, we investigated the function of lncRNAs during the differentiation of human retinal pigment epithelial (ARPE-19) cells, identifying LINC00276 as being consistently and robustly upregulated during RPE differentiation. Silencing LINC00276-202 (ENST00000418420.1) using antisense GapmeRs significantly reduced the expression of RPE differentiation-associated genes, including RPE65 , MITF , TRPM1 , and TRPM3 , whereas LINC00276-202 overexpression restored their expression levels. In silico analysis identified a shared miR-204-5p/miR-211-5p-responsive region within LINC00276-202 . Both miRNAs produced modest increases in LINC00276-202 -associated reporter activity, whereas deletion of the shared seed-recognition region attenuated this response. Importantly, despite increased accumulation of the seed-deletion transcript, the mutant construct failed to induce RPE65 expression to the extent observed with WT LINC00276-202 , indicating that sequence integrity contributes to its biological activity. RNA pull-down followed by mass spectrometry identified PURB, SART3, ANXA6, and hnRNPL as potential protein binding partners. Of these, hnRNPL was validated as a LINC00276-202 -interacting protein by RNA immunoprecipitation and immunoblotting. LINC00276-202 modulated hnRNPL protein levels, and hnRNPL contributed to LINC00276-202 -mediated regulation of β-catenin protein abundance and TCF/LEF transcriptional activity. Together, these findings identify LINC00276-202 as a novel regulator of RPE differentiation involving a miR-204/miR-211-responsive region, hnRNPL interaction, and β-catenin-associated signaling, with potential relevance to retinal degenerative disease.