## 3 Discussion
Under these conditions, we detected a truncated 16-kDa protein from the G56R-CRISPR cDNA, which corresponded to the predicted truncation of NR2E3 within the DBD. Furthermore, this truncated protein showed a defective localization as compared to WT NR2E3 and even when compared to the mutated G56R NR2E3 protein. More specifically, G56R-CRISPRR2 NR2E3 expression was diffused throughout the cytosol, whereas WT NR2E3 expression was restricted to the (peri)nuclear region. Therefore, even if G56R-CRISPRR escaped NMD in the photoreceptors, its altered structure and mis-localization would most likely lead to its degradation [43]. The removal of a nuclear localization signal (NLS) would be a plausible explanation for the cytoplasmic localization of G56R-CRISPR NR2E3. An in silico search of the 410 aa NR2E3 protein sequence (NP\({}_{-0}\)55064.1) for an NLS using the database cNLS mapper [44], identified a putative, but low-score, bipartite NLS at aa position 69 in the DBD. Consistently, other members of the Nuclear Receptor family are known to contain NLS in the DBD, in the hinge region, or even the LBD [45,46]. The predicted NLS in the NR2E3 DBD is missing from the truncated G56R-CRISPRR proteins, which could account for the lack of nuclear targeting. By contrast, the NLS is unaffected by the G56R variant, which is consistent with the nuclear localization of this mutant protein. What was particularly interesting from this study was the mixed nuclear and perinuclear staining for WT NR2E3, which was a striking contrast from the predominant nuclear staining of the G56R mutant. These results could suggest that NR2E3 shuttles between the nucleus and the cytoplasm to accomplish its roles in the cell. This would not be surprising, as many nuclear receptors are known to reside in the cytoplasm [46], but, until now, it has not been described for NR2E3. Different mechanisms have been reported for the export of nuclear receptors, including nuclear export signals (NES), protein-protein interactions, and posttranslational modifications [47]. The DBD has been reported to act as a NES for many nuclear receptors, such as the Retinoic X receptor, which belongs to the same subfamily as NR2E3 [47]. Thus, we could hypothesize that this is also the case for NR2E3 and that the G56R mutation, which is in the DBD, could directly affect its nuclear export ability. We also screened the NR2E3 sequence for a potential NES while using the LocNES prediction tool [48]. NES were predicted from aa positions 359 to 381 within the LBD, with the highest scoring NES at position 366-380. However, it is difficult to understand how G56R could disrupt this C-terminal signal. A previous study reported the subcellular localization of a panel of \(NR2E3\) variants, including G56R [16]. The studied variants were scored according to a nuclear or cytoplasmic localization and, consistent with our data, G56R was reported to be nuclear. Although there was no strict correlation between the position (DBD versus LBD) of the variants studied and the resulting localization profile, it can be said that the majority of variants resulting in a nuclear localization were situated upstream of aa position 232, and the majority resulting in a cytoplasmic (or mixed) localization were situated downstream (i.e., in the LBD). This could be consistent with the predicted presence of the NES with the LBD of NR2E3 at aa position 366-380. In contrast to our observations, this previous study described a nuclear localization for wild type NR2E3. This is surprising, as there is no doubt as to the perinuclear NR2E3 staining in our study. The cell lines that were used in the two studies were similar (COS1 versus COS7 here), so it is unlikely that this underlies the differences observed. However, the previous study [16] used a pcDNA4 His/Max expression system, which fused a 4-kDa Xpress epitope to the NR2E3 protein. Thus, it is possible that the tag partially interfered with the intracellular trafficking/localization of NR2E3.