SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes

Abstract

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal the link between splicing machinery components with the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome – the most common cause of deaf-blindness. SANS functions has been associated with cytoplasmic processes so far. Here, we show SANS localization in Cajal bodies and nuclear speckles. There SANS interacts with components of spliceosomal complexes and the large splicing cofactor SON and PRPFs of the tri-snRNP complex. SANS is required for the release of tri-snRNPs from Cajal bodies and their recruitment to nuclear speckles. SANS depletion alters spliceosome assembly kinetics, leading to stalled complex A formation, which can be chased to spliced products by the addition of tri-snRNPs. SANS deficiency and USH1G mutations affects splicing of genes related to cell proliferation and USH. We provide the first evidence that splicing deregulation may participate at the pathophysiology of Usher syndrome.

Expression and subcellular localization of USH1C/harmonin in the human retina provide insights into pathomechanisms and therapy

“…However, USH1C/harmonin has not been identified as a target for the splicing control machinery of Musashi proteins. Recently, we were able to show that the USHG1 protein SANS interacts with core components of the spliceosome and regulates splicing including constitutive and alternative splicing of USH1C/harmonin (57). In particular, the latter study shows that SANS causes the retention of exon 11, thereby leading to alternative expression of the human harmonin_a1 and _a4 transcripts.…”

“…In particular, the latter study show that SANS causes the retention of exon 11 and thus the alternative expression of the human harmonin a1 and a4 transcripts. Future studies are important and necessary to understand the interplay and interference of the two USH1 proteins SANS and harmonin in the retina at two different levels: in common protein complexes (SANS-SAM/PBM binding to the Ndomain and PDZ3 of harmonin), possibly related to transport processes (Adato et al, 2005;Maerker et al, 2008;Yan et al, 2010), and during splicing, where SANS presumably can interfere with USH1C/harmonin pre-mRNA (Yildirim et al, 2021).…”

Progressive protein aggregation in PRPF31 patient retinal pigment epithelium cells: the mechanism and its reversal through activation of autophagy

“…The assembly and maturation of tri-snRNPs occur in Cajal bodies, and defects in the assembly or release of tri-snRNPs results in the accumulation of incomplete snRNPs in Cajal bodies and consequently stalling of spliceosomes at complex A (15,22). Thus, we performed RNA-FISH using fluorescent probes against U4, U6 and U5 snRNAs followed by immunostaining for coilin, a Cajal body marker.…”

“…Nonetheless, a detailed analysis of the impacts of PRPF mutations on tri-snRNPs formation within Cajal bodies of retinal cells, spliceosome assembly in nuclear speckles and formation of active spliceosomes directly on patient specific RPE and retinal cells has not been reported previously. Using a combination of RNA-FISH and immunofluorescence microscopy, we demonstrate a significant defect in the tri-snRNP assembly in patient photoreceptor cells leading to a decrease in U4/U6 di-snRNPs and accumulation of U5 snRNP in Cajal bodies, the nuclear membrane-less organelles where tri-snRNPs are assembled and fully matured (15). Strikingly, glycerol gradient fractionation combined with Northern and Western blotting, demonstrated for the first time that the mutant PRPF31 is not incorporated into the spliceosomal complexes in RP11-RPE cells.…”

“…Fluorescence in situ hybridisation (FISH) was performed using probes labelled with Alexa 647 at the 5'-end. The sequences of probes and the RNA-FISH method were as previously described (15,16). After FISH, cells were immunostained for coilin and prepared for confocal microscopy as described above.…”