Abstract
Purpose :
Human Usher syndrome (USH) is the most common form of hereditary deaf-blindness, characterized by inner ear defects and vision loss. To date, there is no treatment for the ocular phenotype of any USH subtype, mainly as pathomechanisms of the disease in the eye are far from being understood. We aim to elucidate the USH pathophysiology by characterizing the function of USH proteins and pathways related to them. We have recently shown that the USH1G protein SANS also plays a role in pre-mRNA splicing in the nucleus. Here, we focus on the identification of splicing events in target genes altered in cell and animal models.
Methods :
Transcriptome analyses of the Ush1g/sans knockout (ko) retinas compared to the appropriate controls were performed via RNAseq. Differential splicing events were identified and quantified by JUM analysis. Altered splicing events were validated by capillary electrophoresis and in vitro mini gene-splicing assays in cells. Affected genes were related to functional modules by GO analysis (R-package clusterProfiler).
Results :
Out of 16,678 events, 272 events were significantly altered in the retina of ko versus wt retinas. The majority of these events corresponded to intron retentions (∼30%) followed by composite events and cassette exons (both ∼ 20%), alternative 5’ splice sites, and alternative 3’ splice sites. GO analysis revealed that the splicing of genes of retina-specific modules such as the phototransduction or the photoreceptor cilium was mostly affected by the absence of Ush1g/sans. Interestingly, these target genes included many USH genes (8 out of 10). These splicing defects also pass validation in USH1G patient-derived cells.
Conclusions :
Our results show that USH1G/SANS participates in the regulation of pre-mRNA splicing of retinal genes. Furthermore, our data suggest that pathogenic mutations in USH1G lead to splicing defects in retinal genes, particularly USH genes. We found evidence that the pathophysiology of USH1G is due to defective splicing of retinal genes, resulting in defective gene products of other retinal genes that underlie the retinal degeneration in USH1G.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.