Abstract
Purpose :
The human Usher syndrome (USH) is the most common form of inherited deaf-blindness. Here, we aimed to decipher the molecular function of the USH1G protein SANS to gain insights into the pathophysiology underlying USH and to identify novel therapeutic targets.
Methods :
We searched for potential interaction partners of SANS by screening yeast-2-hybrid retinal cDNA libraries and affinity proteomics. We validated identified putative interactions by complementary protein-protein interaction experiments and determined the subcellular localization of interacting partners by immunocytochemistry (ICC) and in situ proximity ligation assays (PLAs). To elucidate the role of SANS in the splicing process, we tested the effect of siRNA-mediated depletion of USH1G on splicing by in-vitro splicing assays, RNA-FISH and Northern blots.
Results :
We identified and validated the interaction of SANS with several splicing related molecules and pre-RNA splicing factors (PRPFs), namely SF3B1, PRPF6, PRPF31, hSnu114, Matrin3, and PRPF38. We showed that SANS co-localized with spliceosome components in nuclear speckles, the splicing compartment of the nucleus. siRNA-mediated depletion revealed that SANS is essential for defined splicing events in 19 test genes as well as USH1C and MYO7A (USH1B) in HEK293T cells. Capillary electrophoresis of RT-PCR amplicons further revealed similar splicing perturbation profiles in PRPFs, SF3B1 and SANS depleted cells. In addition, ICC, RNA-FISH, in vitro splicing assays and Northern blot analyses revealed a crucial role of SANS in maturation of the U4/U6-U5 tri-snRNP complex in Cajal bodies and the formation of the spliceosomal complex B.
Conclusions :
We show that SANS is a component of splicing complexes and cooperates in the regulation of splicing. Depletion of SANS cause apparent splicing of genes related to the human Usher syndrome. Thereby, we provide evidence that deregulation of splicing can underlay the pathophysiology related to the Usher syndrome and other retinal ciliopathies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.