Molecular Mechanisms in Retinoschisin (RS1) Signal Sequence Variants Associated With X-Linked Retinoschisis (XLRS)

Abstract

Purpose: : Signal sequence is a short N-terminal peptide (4-60 amino acids long) that targets a secreted protein such as RS1 through the cell membrane. Signal sequence variants of RS1 are associated with XLRS. We studied the molecular mechanisms underlying the signal sequence mutations p.M1L, p.L12H, p.L13P and a newly identified c.52G>A (IVS1-1 G >A) mutation in the terminal nucleotide of exon1.

Methods: : RS1 cDNA was amplified from human retina total RNA by RT-PCR and cloned into the pCR II-TOPO vector by standard methods. Substitution mutations p. M1L, p. L12H and p. L13P were introduced into the RS1 cDNA by site-directed mutagenesis. A synthetic RS1 minigene encoding genomic introns 1 & 2 in frame with exons 1-6 was constructed in two cloning steps with or without G >A mutation in the terminal nucleotide of exon 1 which is within the signal sequence. The biochemical phenotypes of the mutants were analyzed by (a) transient transfection of WT & mutant plasmid DNA into COS-7cells followed by the analysis of (b) RNA by RT-PCR and DNA sequencing and (c) RS1 protein byimmunoblotting.

Results: : Each of these four point mutations in the signal sequence motif affected one of the steps in RS1 biosynthesis, namely post-transcriptional pre-RNA splicing (c.52G>A), initiation of protein translation (p. M1L) and the maturation of the nascent protein (p. L12H, p. L13P). The mutation c.52G>A resulted in several biochemical phenotypes: approximately 90% of RNA was lacking exon 2 or exon 1 & 2 and only 10% was normally spliced RS1 mRNA.

Conclusions: : These four signal sequence variants contribute to a null-protein biochemical phenotype due to the instability of mRNA (nonsense mediated mRNA decay) or rapid degradation of the mutant protein by the proteasomal pathway. The clinical severity of the disease in these families ranged from severe (in the middle aged brothers with p.M1L) to more modest (p.L13P and c.52G>A).

Keywords: retinal degenerations: hereditary • retinal degenerations: cell biology • retina