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

Abstract

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. FASEB J. 33, 9422–9433 (2019). http://www.fasebj.org

Dissecting the role of EYS in retinal degeneration: clinical and molecular aspects and its implications for future therapy

“…Proteome dynamic regulation via splicing is conserved in vertebrate neurons and contributes to the nervous system's complexity [ 34 ]. Concerning EYS, a growing number of variant transcripts have been identified, resulting from alternative splicing [ 35 , 36 ]. Expression of different isoforms has been detected in the retina and other tissues.…”

Systematic detection of Mendelian and non-Mendelian variants associated with retinitis pigmentosa by genome-wide association study

“…More specifically, Peak 1 (rs76960013, allele frequency (AF) = 0.0414, odds ratio (OR) = 3.95, P = 1.18x10 -13 ) was in linkage disequilibrium (LD; r 2 = 0.68) with c.2528G>A (p.G843E; hereafter termed G843E; Table 1). G843E with an AF (0.0171) unusually high for ARRP has been described in conflicting ways in past reports, as having uncertain significance 23 , being non-pathogenic 24 , possibly being pathogenic (although without sufficient supporting evidence) 25 , and was unreported in the two largest genetic screening projects targeting Japanese RP patients 9,11 .…”

“…Second, we established patient-derived lymphoblasts from homozygotes of G843E and S1653Kfs and studied the expression of EYS mRNA by forced transcription of EYS through insertion of a constitutively active CAG promoter immediately upstream of the initiation codon of the gene ( Figure S3) 15 . Among the seven main transcript variants reported 25 , the retina-specific long isoforms (transcription variants 1 and 4) are considered essential for photoreceptor biology 25,26 . RT-PCR followed by Sanger sequencing indicated that, mRNA containing G843E was expressed without the loss of the C-terminal end of the retina-specific long isoform (Figure 2A and B).…”

“…Exon numbers are based on Tv1. Note, Tv5 was identified only in fibroblasts25 . B. RT-PCR analysis of EYS expression.…”

“…Expression analysis of the EYS G843E mutant allele in patient-derived lymphoblastoid cell lines A. A schematic map of the RT-PCR primer designed in relation to the exon-intron structure and mutations (G843E and S1653Kfs) in EYS and published transcript variants (Tv) 25. The locations of G843E (Exon 16) and S1653Kfs (Exon 26) are indicated by the arrows.…”