Abstract
Purpose: :
NRL is the key transcription factor, essential and sufficient, for rod differentiation. NRLis a member of the Maf transcription factor family and its activity is regulated by post-translational modifications. We have demonstrated that multiple phosphorylated isoforms of NRL are expressed in rods. This study was undertaken to investigate the role of SUMOylation in the regulation of NRL biological activity.
Methods: :
SUMOplot analysis was used to predict potential SUMOylation sites within the NRL protein sequence. SUMOylation of NRL was investigated in an in vitro SUMOylation assay using purified NRL-GST protein and by immunoprecipitation from transfected HEK293 cells and mouse retina. Different SUMOylation mutants of NRL were generated by site-directed mutagenesis. Their transcriptional activity was assessed in HEK293 cells using luciferase reporter activity assays. NRL mutants were electroporated into the retina of newborn Nrl-/- mice, and differentiation of cones and rods was assessed by immunohistochemistry after 21 days.
Results: :
SUMOplot analysis predicted two lysine residues at position 20 and 24 of NRL as potential sites for SUMOylation. We demonstrate that NRL is indeed SUMOylated both in vitro and in vivo. The molecular weight of SUMOylated NRL is determined to be around 50 kDa, suggesting that 2 SUMO-1 proteins are linked to NRL. NRL-K20R, NRL-K24R, and NRL-K20R/K24R mutants showed lower transcriptional activity when co-expressed with CRX on rhodopsin and NR2E3 promoters. Overexpression of the NRL-K20R/K24R mutant in Nrl-/- mice led to photoreceptors expressing both rod and cone specific marker proteins, instead of rods or cones alone.
Conclusions: :
Our results show that SUMOylation controls NRL activity and is critical for appropriate development of rod photoreceptors.
Keywords: transcription factors • retinal development • photoreceptors