Intra- and interspecies comparison of EYS transcripts highlights its characteristics in the eye.

Inherited mutations in the eyes shut homolog (EYS) gene cause retinitis pigmentosa. Although knock out of eys in zebrafish is pathogenic, the molecular function of EYS in vertebrate photoreceptors is poorly understood. Here, we show that the 5' portion of EYS is eye-specific across vertebrates. We previously determined that a 3' fragment of EYS with an unknown transcription start site is expressed in human dermal fibroblasts (HDF). To obtain insights into the molecular function of EYS in vertebrate photoreceptors, we extensively analyzed EYS (eys) expression in the human fibroblast cell line HDF-adult (HDF-a), the Y79 retinoblastoma cell line, and in zebrafish eyes using rapid amplification of cDNA end, cap analysis of gene expression, RNA sequencing, and RT-PCR. In HDF-a cells, we identified a novel transcript variant (tv), tv5, transcribed from exon 37. In Y79 cells and zebrafish eyes, EYS (eys) was predominantly transcribed from exon 1 or 2, whereas it was transcribed exclusively from exon 37 in HDF-a cells. In the zebrafish eye, there were splice variants that introduced stop codons, resulting in complete loss of the 3' portion of the RNA. These comparative approaches indicate that the 5' portion of the EYS (eys) mRNA appears to be photoreceptor-specific and that the compositions of the deduced EYS proteins in the eye are well-conserved across vertebrates.-Takita, S., Miyamoto-Matsui, K., Seko, Y. Intra- and interspecies comparison of EYS transcripts highlights its characteristics in the eye.

The manner of decay of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients depends on the type of mutation.

BACKGROUND: Generation of induced photoreceptors holds promise for in vitro modeling of intractable retinal diseases. Retinitis pigmentosa is an inherited retinal dystrophy that leads to visual impairment. The EYS gene was reported to be the most common gene responsible for autosomal recessive retinitis pigmentosa (arRP). arRP with defects in the EYS gene is denoted by "EYS-RP". We previously established a "redirect differentiation" method to generate photosensitive photoreceptor-like cells from commercially available human dermal fibroblasts. In this study, we produced photoreceptor-like cells from dermal fibroblasts of EYS-RP patients as a replacement for the degenerative retinas using "redirect differentiation". We analyzed defective transcripts of the EYS gene in these cells to elucidate phenotypes of EYS-RP patients because decay of transcripts was previously suggested to be involved in phenotypic variation associated with diseases. METHODS: Using "redirect differentiation" by CRX, RAX, NeuroD and OTX2, we made photoreceptor-directed fibroblasts derived from three normal volunteers and three EYS-RP patients with homozygous or heterozygous mutations. We tested inducible expression of the photoreceptor-specific genes (blue opsin, rhodopsin, recoverin, S-antigen, PDE6C) in these cells. We then analyzed transcripts derived from three different types of the defective EYS gene, c.1211dupA, c.4957dupA and c.8805C > A, expressed in these cells by RT-PCR and sequencing. RESULTS: Photoreceptor-specific genes including the EYS gene were up-regulated in all the photoreceptor-directed fibroblasts tested. However, expression levels of defective transcripts were markedly different depending on the type of mutation. Transcripts derived from these three defective genes were scarcely detected, expressed at a lower level, and expressed at almost the same level as in normal volunteers, respectively. CONCLUSIONS: Expression levels of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts of EYS-RP patients vary depending on the type of mutation. Variation in expression levels in transcripts having c.1211dupA, c.4957dupA and c.8805C > A suggests that almost complete nonsense-mediated mRNA decay (NMD), partial NMD and escape from NMD occurred for these transcripts, respectively. To determine the relationship with phenotypic variations in EYS-RP patients, more samples are needed. The present study also suggests that the redirect differentiation method could be a valuable tool for disease modeling despite some limitations.