Abstract
Purpose :
Emerging evidence indicates that some non-coding RNA molecules may harbor short open reading frames (sORFs) that code for functional micropeptides that may play a major role in regulating many pathophysiological processes. However, the functions of these remain largely unexplored given their small size. sORFs can act independently as ligands or signaling molecules by engaging with and modulating larger regulatory proteins to fine-tune complex biological systems. In this study, we attempt to identify potential protein-coding sORFs from LINC00276, a non-coding RNA identified in differentiated ARPE-19 cells.
Methods :
Bioinformatic analyses based on evolutionary conservation were used to identify sORFs with the potential to encode conserved micropeptides. Also, we employed the Coding Potential Calculator version 2 (CPC2) algorithm, a novel discriminative algorithm assessing sequence intrinsic features at the DNA/RNA level, to predict high-quality sORFs. The identified sORFs were then cloned into pEGFP-N1 vector in-frame with C-terminus GFP and 6xHis tag. The expression of these constructs in ARPE-19 cells was analyzed by immunofluorescence and western blotting after transfection.
Results :
In a screen of LINC00276 transcripts, we identified an evolutionarily conserved sORF with the potential to encode a highly conserved 19 amino acid (aa) micropeptide located in exon 2, which is common to both LINC00276 transcripts. Using CPC2, we identified another sORF encoding a 74 aa peptide located in exon 4, only present in the second transcript of LINC00276. Expression of these constructs in ARPE-19 cells yielded peptides corresponding to the predicted molecular weight of the fusion peptides, detected by western blot. Both micropeptides, of 19 and 74 aa, showed GFP expression but a distinct GFP expression pattern was observed with the 74 aa micropeptide in ARPE-19 cells.
Conclusions :
In this study, we provide evidence for the presence of two putative micropeptides encoded in LINC00276. These may play a role in modulating the expression of genes associated with RPE differentiation. Further studies on the identification and functional characterization of the micropeptides are required to elucidate their biological functions and may provide further insight into the cellular role of LINC00276 in regulating RPE characteristics.
This is a 2021 ARVO Annual Meeting abstract.