Abstract
Purpose :
Rod-derived cone viability factor (RdCVF) is a rod-derived protein that enables cone photoreceptor survival by binding to BSG1/GLUT1 and stimulating aerobic glycolysis. It is normally produced solely in rods from an unspliced (i.e., intron-retaining) isoform of nucleoredoxin-like 1 (NXNL1) RNA. We recently found that the RNA binding protein nucleolin (NCL) binds specifically to a conserved RNA secondary structure located in the NXNL1 intron suggesting a possible role in NXNL1 intron retention and RdCVF expression. Consistent with this possibility, in the primate retina, rods localized in the periphery of the outer retina express NCL and both the spliced and the unspliced NXNL1 gene products, while the cones at the fovea lack NCL and express only the spliced NXNL1 product, RdCVFL. Whereas NCL and RdCVF are normally expressed solely in rods, both NCL and RdCVF are expressed in retinoblastoma cells that derive from cone precursors following biallelic inactivation of the RB1 gene. Here, we examine whether NCL triggers intron retention and RdCVF expression in retinoblastoma cells.
Methods :
After identifying the optimal infection condition with low cytotoxicity, several retinoblastoma cell lines were transduced with NCL shRNAs via lentivirus infection in order to inhibit NCL expression. After that, NCL and RdCVF expression are quantified by RT-PCR.
Results :
In three retinoblastoma cell lines with low-, middle-, and high-level chromosomal copy number alterations, the expression of RdCVF and NCL mRNAs is significantly higher than in the primate macula enriched in cone photoreceptors. In these transformed cells we will determine if shRNA-mediated decrease in NCL expression causes decreased NXNL1 intron retention and decreased RdCVF expression.
Conclusions :
In retinoblastoma cells, high-level expression of NCL is associated with the expression of the intron retaining NXNL1 mRNA and production of RdCVF protein, as normally occurs in rods. Accordingly, RdCVF could stimulate aerobic glycolysis in an autocrine manner to enable the proliferation of retinoblastoma tumor independently of rod trophic support.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.