Stable Intronic Sequences and Exon Skipping Events in the Human RPE65 Gene: Analysis of Expression in Retinal Pigment Epithelium Cells and Cell Culture Models.

Olga Postnikova,Nady Golestaneh,Igor B Rogozin,Eugenia Poliakov,T Michael Redmond

doi:10.3389/fgene.2019.00634

Abstract

Currently, there is much interest in intronic sequence-containing long non-coding RNAs and the role of intronic transcription in regulation of cellular metabolism and fate. Several stable intronic sequence RNAs (sisRNAs) were recently implicated in regulation of parental genes. To investigate transcription from introns of the RPE65 gene, we analyzed RNA-seq and Nanopore sequencing data from different cell models of human retinal pigment epithelium (RPE) and native bovine RPE. We discovered putative stable poly-adenylated transcripts with sequences corresponding to intronic regions of the RPE65 gene in the cytoplasm of RPE cells. These stable intronic sequences could be important for RPE65 transcription, splicing or translation. We also analyzed alternative splicing events in RPE65. Frequent exon skipping events involving exons 2, 3, and 7 were detected. The rate of these events was much higher in human RPE cell cultures compared with native RPE , consistent with lack of translation of RPE65 mRNA in cell cultures.

Highlights

Light activation of visual pigments in the mammalian retina leads to the phototransduction pathway and concurrent release of the photoisomerized chromophore
We found several complete or nearly complete RPE65 mRNA sequences and many incomplete 3′untranslated region (UTR) regions (Supplementary Table S1)
Mapping of RNA-seq reads to the RPE65 gene suggested that 4M_ARPE-19 and human retinal pigment epithelium (RPE)/choroid (Whitmore et al, 2014) samples are similar the level of expression is much lower in 4M_ARPE-19: the number of raw reads in each position is roughly 100 times lower with approximately similar sizes of RNA-seq data sets (Figure 1 and Table 1)

Summary

Introduction

Light activation of visual pigments in the mammalian retina leads to the phototransduction pathway and concurrent release of the photoisomerized chromophore. Mammalian vision depends on constant recycling of 11-cis retinal chromophore in the multistep visual (retinoid) cycle in the retinal pigment epithelium (RPE), a monolayer epithelium adjacent to the photoreceptor outer segments. RPE65 is the central isomerohydrolase in the RPE visual cycle that catalyzes the conversion of alltrans-retinyl ester (atRE) into 11-cis retinol (Jin et al, 2005; Moiseyev et al, 2005; Redmond et al, 2005). RPE65 is one of three mammalian members of the carotenoid oxygenase family and has evolved to become a retinol isomerohydrolase in the vertebrate retinal visual cycle (Poliakov et al, 2012). Except for one recently described mutation D477G (Bowne et al, 2011), RPE65 mutations have been invariably recessively inherited (Thompson et al, 2000)

Methods

Results

Conclusion