Pathogenic Variants in USH1G/SANS Alter Protein Interaction with Pre-RNA Processing Factors PRPF6 and PRPF31 of the Spliceosome.

Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intranuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH), the most common form of hereditary deaf-blindness. Here, we aim to gain further insights into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by the binding of PRPF6. Furthermore, we find that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs, implying a significance for the USH1G pathophysiology.

USH2A variants causing retinitis pigmentosa or Usher syndrome provoke differential retinal phenotypes in disease-specific organoids.

There is an emblematic clinical and genetic heterogeneity associated with inherited retinal diseases (IRDs). The most common form is retinitis pigmentosa (RP), a rod-cone dystrophy caused by pathogenic variants in over 80 different genes. Further complexifying diagnosis, different variants in individual RP genes can also alter the clinical phenotype. USH2A is the most prevalent gene for autosomal-recessive RP and one of the most challenging because of its large size and, hence, large number of variants. Moreover, USH2A variants give rise to non-syndromic and syndromic RP, known as Usher syndrome (USH) type 2, which is associated with vision and hearing loss. The lack of a clear genotype-phenotype correlation or prognostic models renders diagnosis highly challenging. We report here a long-awaited differential non-syndromic RP and USH phenotype in three human disease-specific models: fibroblasts, induced pluripotent stem cells (iPSCs), and mature iPSC-derived retinal organoids. Moreover, we identified distinct retinal phenotypes in organoids from multiple RP and USH individuals, which were validated by isogenic-corrected controls. Non-syndromic RP organoids showed compromised photoreceptor differentiation, whereas USH organoids showed a striking and unexpected cone phenotype. Furthermore, complementary clinical investigations identified macular atrophy in a high proportion of USH compared with RP individuals, further validating our observations that USH2A variants differentially affect cones. Overall, identification of distinct non-syndromic RP and USH phenotypes in multiple models provides valuable and robust readouts for testing the pathogenicity of USH2A variants as well as the efficacy of therapeutic approaches in complementary cell types.

Usher syndrome proteins ADGRV1 (USH2C) and CIB2 (USH1J) interact and share a common interactome containing TRiC/CCT-BBS chaperonins.

The human Usher syndrome (USH) is the most common form of a sensory hereditary ciliopathy characterized by progressive vision and hearing loss. Mutations in the genes ADGRV1 and CIB2 have been associated with two distinct sub-types of USH, namely, USH2C and USH1J. The proteins encoded by the two genes belong to very distinct protein families: the adhesion G protein-coupled receptor ADGRV1 also known as the very large G protein-coupled receptor 1 (VLGR1) and the Ca2+- and integrin-binding protein 2 (CIB2), respectively. In the absence of tangible knowledge of the molecular function of ADGRV1 and CIB2, pathomechanisms underlying USH2C and USH1J are still unknown. Here, we aimed to enlighten the cellular functions of CIB2 and ADGRV1 by the identification of interacting proteins, a knowledge that is commonly indicative of cellular functions. Applying affinity proteomics by tandem affinity purification in combination with mass spectrometry, we identified novel potential binding partners of the CIB2 protein and compared these with the data set we previously obtained for ADGRV1. Surprisingly, the interactomes of both USH proteins showed a high degree of overlap indicating their integration in common networks, cellular pathways and functional modules which we confirmed by GO term analysis. Validation of protein interactions revealed that ADGRV1 and CIB2 mutually interact. In addition, we showed that the USH proteins also interact with the TRiC/CCT chaperonin complex and the Bardet Biedl syndrome (BBS) chaperonin-like proteins. Immunohistochemistry on retinal sections demonstrated the co-localization of the interacting partners at the photoreceptor cilia, supporting the role of USH proteins ADGRV1 and CIB2 in primary cilia function. The interconnection of protein networks involved in the pathogenesis of both syndromic retinal dystrophies BBS and USH suggest shared pathomechanisms for both syndromes on the molecular level.

Correction: Winkler et al. The Adhesion G-Protein-Coupled Receptor GPR115/ADGRF4 Regulates Epidermal Differentiation and Associates with Cytoskeletal KRT1. Cells 2022, 11, 3151.

In the original publication [...].

The adhesion G protein-coupled receptor VLGR1/ADGRV1 controls autophagy.

VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest known adhesion G protein-coupled receptor. Mutations in VLGR1/ADGRV1 cause Usher syndrome (USH), the most common form of hereditary deaf-blindness, and have been additionally linked to epilepsy. Although VLGR1/ADGRV1 is almost ubiquitously expressed, little is known about the subcellular function and signalling of the VLGR1 protein and thus about mechanisms underlying the development of diseases. Using affinity proteomics, we identified key components of autophagosomes as putative interacting proteins of VLGR1. In addition, whole transcriptome sequencing of the retinae of the Vlgr1/del7TM mouse model revealed altered expression profiles of gene-related autophagy. Monitoring autophagy by immunoblotting and immunocytochemistry of the LC3 and p62 as autophagy marker proteins revealed evoked autophagy in VLGR1-deficient hTERT-RPE1 cells and USH2C patient-derived fibroblasts. Our data demonstrate the molecular and functional interaction of VLGR1 with key components of the autophagy process and point to an essential role of VLGR1 in the regulation of autophagy at internal membranes. The close association of VLGR1 with autophagy helps to explain the pathomechanisms underlying human USH and epilepsy related to VLGR1 defects.

Monitoring paxillin in astrocytes reveals the significance of the adhesion G protein coupled receptor VLGR1/ADGRV1 for focal adhesion assembly.

VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest adhesion G protein-coupled receptor (aGPCR). Mutations in VLGR1/ADGRV1 are associated with human Usher syndrome, the most common form of deaf-blindness, and also with epilepsy in humans and mice. VLGR1 is expressed almost ubiquitously but is mainly found in the CNS and in the sensory cells of the eye and inner ear. Little is known about the pathogenesis of the diseases related to VLGR1. We previously identified VLGR1 as a vital component of focal adhesions (FAs) serving as a metabotropic mechanoreceptor controls cell spreading and migration. FAs are highly dynamic and turnover in response to internal and external signals. Here, we aimed to elucidate how VLGR1 participates in FA turnover. Nocodazole washouts and live cell imaging of paxillin-DsRed2 consistently showed that FA disassembly was not altered, but de novo assembly of FA was significantly delayed in Vlgr1-deficient astrocytes, indicating that VLGR1 is enrolled in FA assembly. In FRAP experiments, recovery rates were significantly reduced in Vlgr1-deficient FAs, indicating reduced turnover kinetics in VLGR1-deficient FAs. We showed that VLGR1 regulates cell migration by controlling the FA turnover during their assembly and expect novel insights into pathomechanisms related to pathogenic dysfunctions of VLGR1.

The Usher syndrome 1C protein harmonin regulates canonical Wnt signaling.

Human Usher syndrome (USH) is the most common form of hereditary combined deaf-blindness. USH is a complex genetic disorder, and the pathomechanisms underlying the disease are far from being understood, especially in the eye and retina. The USH1C gene encodes the scaffold protein harmonin which organizes protein networks due to binary interactions with other proteins, such as all USH proteins. Interestingly, only the retina and inner ear show a disease-related phenotype, although USH1C/harmonin is almost ubiquitously expressed in the human body and upregulated in colorectal cancer. We show that harmonin binds to ß-catenin, the key effector of the canonical Wnt (cWnt) signaling pathway. We also demonstrate the interaction of the scaffold protein USH1C/harmonin with the stabilized acetylated ß-catenin, especially in nuclei. In HEK293T cells, overexpression of USH1C/harmonin significantly reduced cWnt signaling, but a USH1C-R31* mutated form did not. Concordantly, we observed an increase in cWnt signaling in dermal fibroblasts derived from an USH1C R31*/R80Pfs*69 patient compared with healthy donor cells. RNAseq analysis reveals that both the expression of genes related to the cWnt signaling pathway and cWnt target genes were significantly altered in USH1C patient-derived fibroblasts compared to healthy donor cells. Finally, we show that the altered cWnt signaling was reverted in USH1C patient fibroblast cells by the application of Ataluren, a small molecule suitable to induce translational read-through of nonsense mutations, hereby restoring some USH1C expression. Our results demonstrate a cWnt signaling phenotype in USH establishing USH1C/harmonin as a suppressor of the cWnt/ß-catenin pathway.

Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).

The Adhesion G-Protein-Coupled Receptor GPR115/ADGRF4 Regulates Epidermal Differentiation and Associates with Cytoskeletal KRT1.

Among the 33 human adhesion G-protein-coupled receptors (aGPCRs), a unique subfamily of GPCRs, only ADGRF4, encoding GPR115, shows an obvious skin-dominated transcriptomic profile, but its expression and function in skin is largely unknown. Here, we report that GPR115 is present in a small subset of basal and in most suprabasal, noncornified keratinocytes of the stratified epidermis, supporting epidermal transcriptomic data. In psoriatic skin, characterized by hyperproliferation and delayed differentiation, the expression of GPR115 and KRT1/10, the fundamental suprabasal keratin dimer, is delayed. The deletion of ADGRF4 in HaCaT keratinocytes grown in an organotypic mode abrogates KRT1 and reduces keratinocyte stratification, indicating a role of GPR115 in epidermal differentiation. Unexpectedly, endogenous GPR115, which is not glycosylated and is likely not proteolytically processed, localizes intracellularly along KRT1/10-positive keratin filaments in a regular pattern. Our data demonstrate a hitherto unknown function of GPR115 in the regulation of epidermal differentiation and KRT1.

The Adhesion GPCR VLGR1/ADGRV1 Regulates the Ca2+ Homeostasis at Mitochondria-Associated ER Membranes.

The very large G protein-coupled receptor (VLGR1, ADGRV1) is the largest member of the adhesion GPCR family. Mutations in VLGR1 have been associated with the human Usher syndrome (USH), the most common form of inherited deaf-blindness as well as childhood absence epilepsy. VLGR1 was previously found as membrane-membrane adhesion complexes and focal adhesions. Affinity proteomics revealed that in the interactome of VLGR1, molecules are enriched that are associated with both the ER and mitochondria, as well as mitochondria-associated ER membranes (MAMs), a compartment at the contact sites of both organelles. We confirmed the interaction of VLGR1 with key proteins of MAMs by pull-down assays in vitro complemented by in situ proximity ligation assays in cells. Immunocytochemistry by light and electron microscopy demonstrated the localization of VLGR1 in MAMs. The absence of VLGR1 in tissues and cells derived from VLGR1-deficient mouse models resulted in alterations in the MAM architecture and in the dysregulation of the Ca2+ transient from ER to mitochondria. Our data demonstrate the molecular and functional interaction of VLGR1 with components in MAMs and point to an essential role of VLGR1 in the regulation of Ca2+ homeostasis, one of the key functions of MAMs.

Affinity Proteomics Identifies Interaction Partners and Defines Novel Insights into the Function of the Adhesion GPCR VLGR1/ADGRV1.

The very large G-protein-coupled receptor 1 (VLGR1/ADGRV1) is the largest member of the adhesion G-protein-coupled receptor (ADGR) family. Mutations in VLGR1/ADGRV1 cause human Usher syndrome (USH), a form of hereditary deaf-blindness, and have been additionally linked to epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying the development of these diseases are still unknown. Our study aimed to identify novel, previously unknown protein networks associated with VLGR1 in order to describe new functional cellular modules of this receptor. Using affinity proteomics, we have identified numerous new potential binding partners and ligands of VLGR1. Tandem affinity purification hits were functionally grouped based on their Gene Ontology terms and associated with functional cellular modules indicative of functions of VLGR1 in transcriptional regulation, splicing, cell cycle regulation, ciliogenesis, cell adhesion, neuronal development, and retinal maintenance. In addition, we validated the identified protein interactions and pathways in vitro and in situ. Our data provided new insights into possible functions of VLGR1, related to the development of USH and epilepsy, and also suggest a possible role in the development of other neuronal diseases such as Alzheimer's disease.

Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes.

Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.

Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E.

The ARF family of regulatory GTPases is ancient, with 16 members predicted to have been present in the last eukaryotic common ancestor. Our phylogenetic profiling of paralogues in diverse species identified four family members whose presence correlates with that of a cilium/flagellum: ARL3, ARL6, ARL13, and ARL16. No prior evidence links ARL16 to cilia or other cell functions, despite its presence throughout eukaryotes. Deletion of ARL16 in mouse embryonic fibroblasts (MEFs) results in decreased ciliogenesis yet increased ciliary length. We also found Arl16 knockout (KO) in MEFs to alter ciliary protein content, including loss of ARL13B, ARL3, INPP5E, and the IFT-A core component IFT140. Instead, both INPP5E and IFT140 accumulate at the Golgi in Arl16 KO lines, while other intraflagellar transport (IFT) proteins do not, suggesting a specific defect in traffic from Golgi to cilia. We propose that ARL16 regulates a Golgi-cilia traffic pathway and is required specifically in the export of IFT140 and INPP5E from the Golgi.

The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and cilia to regulate ciliogenesis and ciliary protein traffic.

ELMODs are a family of three mammalian paralogues that display GTPase-activating protein (GAP) activity toward a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogues ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing the determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon activating mutant expression of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions.

Isolation and culturing of primary mouse astrocytes for the analysis of focal adhesion dynamics.

Primary astrocytes have gained attention as an important model for in vitro biological and biochemical research in the last decades. In this protocol, we describe a fast and cost-effective technique for isolating, culturing, and maintaining primary mouse astrocytes at ∼ 80% purity levels, which can be used in in vitro studies for migration and focal adhesion dynamics. In addition, we present an optimized transfection and manual quantification approach for focal adhesion analysis in fixed and living cells. For complete details on the use and execution of this protocol, please refer to Kusuluri et al. (2021).

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

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome-the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.

Adhesion G protein-coupled receptor VLGR1/ADGRV1 regulates cell spreading and migration by mechanosensing at focal adhesions.

VLGR1 (very large G protein-coupled receptor-1) is by far the largest adhesion G protein-coupled receptor in humans. Homozygous pathologic variants of VLGR1 cause hereditary deaf blindness in Usher syndrome 2C and haploinsufficiency of VLGR1 is associated with epilepsy. However, its molecular function remains elusive. Herein, we used affinity proteomics to identify many components of focal adhesions (FAs) in the VLGR1 interactome. VLGR1 is localized in FAs and assembles in FA protein complexes in situ. Depletion or loss of VLGR1 decreases the number and length of FAs in hTERT-RPE1 cells and in astrocytes of Vlgr1 mutant mice. VLGR1 depletion reduces cell spread and migration kinetics as well as the response to mechanical stretch characterizing VLGR1 as a metabotropic mechanosensor in FAs. Our data reveal a critical role of VLGR1 in the FA function and enlighten potential pathomechanisms in diseases related to VLGR1.

Roles for ELMOD2 and Rootletin in ciliogenesis.

ELMOD2 is a GTPase-activating protein with uniquely broad specificity for ARF family GTPases. We previously showed that it acts with ARL2 in mitochondrial fusion and microtubule stability and with ARF6 during cytokinesis. Mouse embryonic fibroblasts deleted for ELMOD2 also displayed changes in cilia-related processes including increased ciliation, multiciliation, ciliary morphology, ciliary signaling, centrin accumulation inside cilia, and loss of rootlets at centrosomes with loss of centrosome cohesion. Increasing ARL2 activity or overexpressing Rootletin reversed these defects, revealing close functional links between the three proteins. This was further supported by the findings that deletion of Rootletin yielded similar phenotypes, which were rescued upon increasing ARL2 activity but not ELMOD2 overexpression. Thus, we propose that ARL2, ELMOD2, and Rootletin all act in a common pathway that suppresses spurious ciliation and maintains centrosome cohesion. Screening a number of markers of steps in the ciliation pathway supports a model in which ELMOD2, Rootletin, and ARL2 act downstream of TTBK2 and upstream of CP110 to prevent spurious release of CP110 and to regulate ciliary vesicle docking. These data thus provide evidence supporting roles for ELMOD2, Rootletin, and ARL2 in the regulation of ciliary licensing.

A new mouse model for retinal degeneration due to Fam161a deficiency.

FAM161A mutations are the most common cause of inherited retinal degenerations in Israel. We generated a knockout (KO) mouse model, Fam161atm1b/tm1b, lacking the major exon #3 which was replaced by a construct that include LacZ under the expression of the Fam161a promoter. LacZ staining was evident in ganglion cells, inner and outer nuclear layers and inner and outer-segments of photoreceptors in KO mice. No immunofluorescence staining of Fam161a was evident in the KO retina. Visual acuity and electroretinographic (ERG) responses showed a gradual decrease between the ages of 1 and 8 months. Optical coherence tomography (OCT) showed thinning of the whole retina. Hypoautofluorescence and hyperautofluorescence pigments was observed in retinas of older mice. Histological analysis revealed a progressive degeneration of photoreceptors along time and high-resolution transmission electron microscopy (TEM) analysis showed that photoreceptor outer segment disks were disorganized in a perpendicular orientation and outer segment base was wider and shorter than in WT mice. Molecular degenerative markers, such as microglia and CALPAIN-2, appear already in a 1-month old KO retina. These results indicate that a homozygous Fam161a frameshift mutation affects retinal function and causes retinal degeneration. This model will be used for gene therapy treatment in the future.