Abstract
Purpose: :
LOC387715/ARMS2 is a gene containing 2 exons that has been associated with age-related macular degeneration (AMD) in humans. We have previously shown conservation of both exons at the genomic level within Old World Primates (OWP) and Hominids. However, the acceptor site is not conserved in OWP, and predicted not to promote correct splicing if transcribed. LOC387715/ARMS2 is transcribed in human retinal pigment epithelium (RPE) and placenta. Whether LOC387715/ARMS2 is transcribed and translated in nonhuman primates is unknown. The purpose of this study is to functionally characterize the LOC387715/ARMS2 gene in Macaca mulatta (rhesus macaque).
Methods: :
Total RNA was isolated from M. mulatta neural retina, RPE and placenta (RNAqueous kit, Ambion) and processed by reverse transcriptase (RT) PCR with oligo-dT primers. First-strand cDNA was amplified using forward primers situated in exon 1 and reverse primers within exon 2. PCR products were visualized in an agarose gel under UV light, excised, purified and sequenced. Full length cDNA spanning the predicted initiation codon to the termination codon was cloned into an expression vector.
Results: :
RT-PCR using exon-spanning primers yielded products, of the predicted size for a transcribed and spliced gene, from RPE, neural retina and placenta. Sequence analysis confirmed the presence of an exon 1 donor splice site and is identical to human. However, M. mulatta utilizes an alternative exon 2 acceptor splice site 18bp downstream of the human equivalent, resulting in 11 different codons in exon 2 including an in-frame termination codon. Exon 1 has an in-frame deletion resulting in loss of codon 37. This corresponds to a 330bp open reading frame (ORF) mRNA transcript, from the predicted GUG start codon to the UAA termination codon. Human and rhesus predicted coding region of exon 1 share 90% identity. The 32bp ORF of rhesus exon 2 (codons 100-109) aligns with 93% identity to human. However, this alignment corresponds to codons 106-108 and 24bp of the 3’UTR of human LOC387715/ARMS2 mRNA.
Conclusions: :
We demonstrate that LOC387715/ARMS2 gene is transcribed in retinal tissue in an Old World monkey, a primate that also manifests an AMD phenotype (ARVO 2007 5094/B242) similar to humans. The mRNA is predicted to encode for a 109 amino acid protein. Whether this mRNA is translated is of interest.
Keywords: age-related macular degeneration • gene/expression • retina