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Disruption of RPGR Protein Interaction Network Is the Common Feature of RPGR Missense Variations That Cause XLRP

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Disruption of RPGR Protein Interaction Network Is the Common Feature of RPGR Missense Variations That Cause XLRP Disruption of RPGR protein interaction network is the common feature of RPGR missense variations that cause XLRP Qihong Zhanga,1, Joseph C. Giacaloneb, Charles Searbya, Edwin M. Stoneb,c, Budd A. Tuckerb,c, and Val C. Sheffielda,b,c,1 aDepartment of Pediatrics, University of Iowa, Iowa City, IA 52242; bDepartment of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Iowa City, IA 52242; and cInstitute for Vision Research, University of Iowa, Iowa City, IA 52242 Edited by Joseph C. Besharse, Medical College of Wisconsin, Milwaukee, WI, and accepted by Editorial Board Member Jeremy Nathans December 7, 2018 (received for review October 23, 2018) − Retinitis pigmentosa (RP) is an inherited retinal degenerative gion in the C-terminal domain. The C terminus of the RPGR1 19 disease with severe vision impairment leading to blindness. About protein contains a cluster of basic residues and a consensus prenylation 10–15% of RP cases are caused by mutations in the RPGR gene, site. Rescue experiments in Rpgr knockout mice demonstrate that the with RPGR mutations accounting for 70% of X-linked RP cases. The RPGRORF15 isoform is functionally sufficient to rescue photore- mechanism by which RPGR mutations cause photoreceptor cell ceptor degeneration (13, 14). dysfunction is not well understood. In this study, we show that RPGR localizes to connecting cilia of rod and cone photore- the two isoforms of RPGR (RPGR1−19 and RPGRORF15) interact with 1−19 ceptors, the transitional zone of motile cilia, and primary cilia both endogenous PDE6D, INPP5E, and RPGRIP1L. The RPGR isoform – contains two PDE6D binding sites with the C-terminal prenylation in vivo and in vitro (15 18). RPGR has been shown to be involved site being the predominant PDE6D binding site. The C terminus of in microtubule organization or regulation of transport in primary RPGR1−19 that contains the prenylation site regulates its interac- cilia and actin stability (19–23). Likewise, RPGR is known to in- − tion with PDE6D, INPP5E, and RPGRIP1L. Only the RPGR1 19 iso- teract with numerous proteins including Whirlin (24), Gelsolin (20), form localizes to cilia in cultured RPE1 cells. Missense variations SMC, IFT88, KIF3A, KAP3, NPM1, RAB8A, CEP290, RPGRIP1, GENETICS found in RPGR patients disrupt the interaction between RPGR iso- RPGRIP1L, NPHP4, PDE6D, and INPP5E (17, 21, 22, 25–34). forms and their endogenous interactors INPP5E, PDE6D, and However, the importance of these interactions relative to the RPGRIP1L. We evaluated a RPGR missense variation (M58K) found pathophysiology of RPGR is not known. Numerous mutations in in a family with X-linked retinitis pigmentosa (XLRP) and show RPGR associated with X-linked retinitis pigmentosa (XLRP) have that this missense variation disrupts the interaction of RPGR iso- been identified (35–41). These variants are found across the entire forms with their endogenous interactors. The M58K variation also 1−19 gene, with the majority of mutations occurring in the low complexity disrupts the ciliary localization of the RPGR isoform. Using this ORF15 assay, we also show that some of the RPGR missense variants region of RPGR . How these variants cause XLRP is not well reported in the literature might not actually be disease causing. understood. Here, we show that RPGR missense variations that Our data establishes an in vitro assay that can be used to validate cause XLRP disrupt the interaction of RPGR with its interactors − the potential pathogenicity of RPGR missense variants. and prevent ciliary localization of RPGR1 19. Our study establishes a practical in vitro assay to evaluate RPGR missense variations in RPGR | cilia | retinal degeneration | PDE6D | INPP5E the RLD region in a cell culture system. etinitis pigmentosa (RP) is an inherited retinal degenerative Rdisease that results in the primary loss of rod photoreceptor Significance cells followed by the secondary loss of cone photoreceptor cells (1). The disorder may be nonsyndromic occurring with retina Due to its severity and early onset, X-linked retinitis pigmen- dysfunction alone (1), or syndromic with other neurosensory dis- tosa (XLRP) is a particularly devastating form of retinal de- orders, developmental abnormalities, or complex clinical findings generation. Most males with XLRP come to medical attention such as in Bardet-Biedl syndrome and Usher syndrome (2–4). before the age of 20. Approximately 15% of all RP is X-linked, Mutations in any of dozens of different genes can cause the and more than 70% of these cases are caused by mutations in clinical phenotypes of retinitis pigmentosa, and these mutations the RPGR gene. Currently, there is no known assay to evaluate can be inherited in an autosomal dominant, autosomal recessive, the nature of RPGR missense variants functionally. In this X-linked dominant, X-linked recessive, mitochondrial, or digenic study, we developed an in vitro assay to examine how differ- fashion (5–9). Approximately 15% of all RP is X-linked, and ent missense variations in RPGR cause XLRP. Our assay is based more than 80% of these patients (i.e., more than 12% of the on the RPGR protein interaction network. Our method provides total) have mutations in the gene RPGR (10). About 60% of all a cost-effective test for RPGR functional mutation analysis. RP-causing mutations in RPGR occur in exon 15 of the gene, and Author contributions: Q.Z., E.M.S., B.A.T., and V.C.S. designed research; Q.Z., J.C.G., and two-thirds of these (more than 5% of all RP) occur in a highly C.S. performed research; Q.Z., J.C.G., C.S., and V.C.S. analyzed data; and Q.Z., E.M.S., repetitive region between codons 801 and 1070 (10). B.A.T., and V.C.S. wrote the paper. There are multiple isoforms of RPGR. Two major isoforms of − The authors declare no conflict of interest. RPGR were detected in the retina (11, 12): RPGR1 19, which ORF15 This article is a PNAS Direct Submission. J.C.B. is a guest editor invited by the Editorial has 19 exons encoding an 815-aa protein, and RPGR , Board. which has 15 exons plus part of intron 15 encoding a 1,152-aa Published under the PNAS license. – protein. Both isoforms share exons 1 14. The N-terminal half of 1To whom correspondence may be addressed. Email: [email protected] or val- RPGR protein contains a tandem repeat structure highly similar [email protected]. to the regulator of chromosome condensation (RCC1) named This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. RCC1-like domain (RLD), which regulates the RAN GTPase. 1073/pnas.1817639116/-/DCSupplemental. ORF15 The RPGR protein has a Glu-Gly–rich low complexity re- Published online January 8, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1817639116 PNAS | January 22, 2019 | vol. 116 | no. 4 | 1353–1360 Downloaded by guest on September 25, 2021 Results cannot be targeted to primary cilia (Fig. 1 G–I, also see SI Ap- Both Isoforms of RPGR Interact with Endogenous PDE6D, INPP5E, and pendix, Fig. S2C). 1−19 RPGRIP1L, but only RPGR Localizes to Cilia. Two major RPGR 1−19 isoforms are present in tissues and cells that arise through dif- The C-Terminal of the RPGR Isoform That Contains the Prenylation 1−19 Site Regulates Its Interaction with Endogenous PDE6D, INPP5E, and ferential splicing (SI Appendix, Fig. S1): RPGR and the 1−19 ORF15 RPGRIP1L. The RPGR isoform interacts with PDE6D much retina-specific isoform RPGR (11, 12). It has been shown ORF15 1−19 more strongly than the RPGR isoform, indicating that the that the RPGR isoform interacts with PDE6D, INPP5E, and 1−19 RPGRIP1L and localizes to primary cilia including connecting RPGR isoform has additional binding sites for PDE6D. Altering the prenylation site of cysteine 812 to alanine (Fig. 2 D– cilia of the retina (17, 27, 41). However, it is not known whether − F) or deleting cysteine 812 (Fig. 2 G–I) abolish RPGR1 19 iso- the RPGRORF15 isoform also binds to these interactors. We first ORF15 form ciliary localization, indicating that prenylation of cysteine examined ciliary localization of RPGR by transfecting − 812 is required for RPGR1 19 ciliary localization. Similar results GFP-tagged RPGRORF15 into human retinal pigmented epithe- − were obtained in mouse IMCD3 cells (SI Appendix, Fig. S2). lial (RPE1) cells. In contrast to the RPGR1 19 isoform (Fig. 1 A– Changing the prenylation site of cysteine 812 to alanine or de- C ORF15 D–F ), the RPGR isoform (Fig. 1 ) did not localize to cilia, leting cysteine 812 also greatly decreases but does not fully − instead, it was detected in the cytoplasm. Similar results were abolish RPGR1 19 binding to PDE6D (Fig. 2K), indicating that – − obtained in mouse IMCD3 cells (SI Appendix, Fig. S2 A C). To the C-terminal of the RPGR1 19 is the stronger binding site for determine if the RPGRORF15 can interact with endogenous – PDE6D and that there is another binding site for PDE6D, likely PDE6D, INPP5E, and RPGRIP1L, we generated a Flag-S-tag in the RLD region. Interestingly, mutating the prenylation site of tagged construct and expressed it in HEK293T cells. Using 1−19 ORF15 cysteine 812 to alanine or deleting cysteine 812 increases RPGR pulldown with anti-Flag beads, we show that RPGR also isoform binding to endogenous INPP5E and RPGRIP1L (Fig. 2K). − interacts with endogenous PDE6D, INPP5E, and RPGRIP1L This finding suggests that prenylation of RPGR1 19 modulates its (Fig. 1K). Since both isoforms of RPGR share the RLD region protein structure, which in turn affects the RLD region of the protein, (SI Appendix, Fig. S3), these data suggest that the binding do- the binding region for INPP5E and RPGRIP1L, which leads to − main for PDE6D, INPP5E, and RPGRIP1L resides in the RLD stronger binding of the RPGR1 19 to INPP5E and RPGRIP1L.
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