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Reptin/Ruvbl2 Is a Lrrc6/Seahorse Interactor Essential for Cilia Motility

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Reptin/Ruvbl2 Is a Lrrc6/Seahorse Interactor Essential for Cilia Motility Reptin/Ruvbl2 is a Lrrc6/Seahorse interactor essential for cilia motility Lu Zhao, Shiaulou Yuan1, Ying Cao2, Sowjanya Kallakuri, Yuanyuan Li, Norihito Kishimoto3, Linda DiBella, and Zhaoxia Sun4 Department of Genetics, Yale University School of Medicine, New Haven, CT 06520 Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved June 21, 2013 (received for review January 20, 2013) Primary ciliary dyskinesia (PCD) is an autosomal recessive disease two mutants share nearly identical abnormalities: ventral body caused by defective cilia motility. The identified PCD genes ac- curvature and kidney cysts (22, 23). count for about half of PCD incidences and the underlying mech- Reptin (or Reptin52/Ruvbl2/Ino80J/Tip48) contains an AAA- anisms remain poorly understood. We demonstrate that Reptin/ family ATPase domain resembling the bacterial RuvB, a DNA Ruvbl2, a protein known to be involved in epigenetic and tran- helicase (for a review, see ref. 24). It regulates transcription scriptional regulation, is essential for cilia motility in zebrafish. We through multiple chromatin remodeling complexes (25–28). It further show that Reptin directly interacts with the PCD protein is also found in protein complexes involved in telomere main- Lrrc6/Seahorse and this interaction is critical for the in vivo func- tenance, small nucleolar RNA assembly and DNA damage tion of Lrrc6/Seahorse in zebrafish. Moreover, whereas the expres- responses (29–32). Interestingly, the transcription of reptin is up- sion levels of multiple dynein arm components remain unchanged regulated during flagellum regeneration in Chlamydomonas (33). or become elevated, the density of axonemal dynein arms is re- hi2394 However, the role of Reptin in cilia biology and vertebrate de- duced in reptin mutants. Furthermore, Reptin is highly enriched velopment was rarely studied before this study. in the cytosol and colocalizes with Lrrc6/Seahorse. Combined, In this study, we demonstrate that Reptin is essential for cilia these results suggest that the Reptin-Lrrc6/Seahorse complex is motility in zebrafish. We further show that Reptin directly involved in dynein arm formation. We also show that although interacts with Lrrc6/Seahorse and that this interaction is im- reptinhi2394 the DNA damage response is induced in mutants, it portant for the in vivo function of Lrrc6/Seahorse, indicating that lrrc6/seahorse remains unchanged in cilia biogenesis mutants and reptin functions together with lrrc6/seahorse in a process essential mutants, suggesting that increased DNA damage response is not for cilia motility and is a candidate gene for PCD. We further intrinsic to ciliary defects and that in vertebrate development, fi provide evidence that although the expression levels of multiple Reptin functions in multiple processes, both cilia speci cand dynein arm components are not decreased, the number of dynein cilia independent. arms on the axoneme is reduced in reptin mutants. Moreover, Reptin is highly enriched in the cytosol and colocalizes with ciliopathy | chromatin remodeling complex | dynein arm assembly factor Lrrc6/Seahorse. Combined, these results suggest that the Reptin- Lrrc6/Seahorse complex functions in dynein arm assembly. Ad- lthough the sensory function of the cilium has garnered ditionally, we investigated the level of the DNA damage re- Asignificant attention only in the past decade, defective cilia sponse (DDR) because of its recent association with ciliopathies motility was recognized nearly four decades ago as the cause of (34). Our results show that although DDR is induced in reptin primary (genetic) ciliary dyskinesia (PCD) (1). PCD is a group of mutants, it remains unchanged in both lrrc6/seahorse and the cilia rare human genetic diseases characterized by recurrent infec- biogenesis mutant ift172, suggesting that DDR induction is not tions of the respiratory system, male infertility, and frequently intrinsic to defective cilia and that the Reptin-Lrrc6/Seahorse laterality defects, all of which are tightly linked to cilia motility interaction is not involved in Reptin’s role in DDR. abnormalities. Up to now, 19 PCD genes have been identified (2–20). However, combined they account for about 50% of all Results PCD cases, suggesting the existence of multiple additional causa- Inactivation of reptin Leads to Cilia-Associated Phenotypes. hi2394 tive genes. Despite such significant advances, our understanding was identified in a large scale insertional mutagenesis screen of how cilia motility is regulated is limited and the list of genes conducted in zebrafish (22, 23). The homozygous mutants showed involved in cilia motility remains incomplete. ventral body curvature and kidney cyst (Fig. 1A), whereas het- In motile cilia or flagella, dynein arms are large protein com- erozygous carriers are viable with no obvious phenotypes. Cross- plexes powering cilia motility. Not surprisingly, multiple PCD genes sections verified large cysts in the glomerular–tubular region, as encode dynein arm components, including DNAL1, DNAI1, well as grossly dilated pronephric duct, which was confirmed by DNAI2, DNAH5,andDNAH11 (2–6). In addition, it is understood whole-mount immunostaining using a renal epithelial marker that dynein arm subunits are preassembled in the cytosol, trans- ported into cilia/flagellum, and docked onto the axoneme, although the underlying mechanisms are poorly understood (21). In recent Author contributions: L.Z. and Z.S. designed research; L.Z., S.Y., Y.C., S.K., Y.L., N.K., and years, three PCD genes, i.e., DNAAF1/LRRC50 (7, 8), DNAAF2/ L.D. performed research; L.Z. and S.Y. analyzed data; and L.Z. and Z.S. wrote the paper. fl BIOLOGY KTU (9), and DNAAF3/PF22 (10) have been shown to be involved The authors declare no con ict of interest. in the assembly of dynein arm subunits, highlighting the relevance This article is a PNAS Direct Submission. DEVELOPMENTAL 1 and importance of this process in cilia motility and PCD. Present address: Department of Pediatrics, Yale University School of Medicine, New Very recently, mutations in human LRRC6/SEAHORSE were Haven, CT 06520. 2Present address: School of Life Sciences and Technology, Tongji University, Shanghai linked to PCD (18). Similar to the three known dynein arm as- 20092, People’s Republic of China. sembly factors, mutations in LRRC6/SEAHORSE lead to the 3Present address: Department of Developmental and Regenerative Biology, Nagoya City absence of both the outer and the inner dynein arms, suggesting University, Nagoya 467-8601, Japan. that this might be an additional dynein arm assembly factor (18). 4To whom correspondence should be addressed. E-mail: [email protected]. fi Strikingly, lrrc6/seahorse mutants were previously identi ed in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a genetic screen in zebrafish together with reptin mutants and the 1073/pnas.1300968110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1300968110 PNAS | July 30, 2013 | vol. 110 | no. 31 | 12697–12702 Downloaded by guest on September 25, 2021 A B embryos injected with 400 pg reptin MO displayed similar phe- WT rep notypes as hi2394 mutants, including ventral body curvature and reptin 5’ kidney cysts (Fig. S1C). Further, we introduced a 5-nucleotide WT mismatch into a reptin cDNA clone so that the encoded mRNA reptin 3’ was no longer a target of reptin MO. Coinjection of the mis- matched mRNA rescued the morphant (Fig. S1D). Finally, we actin tested whether expression of reptin could rescue hi2394 mutants. rep WT rep Specifically, we injected 150 pg reptin-egfp mRNA or as a control, 150 pg myc-egfp mRNA into embryos from crosses of heterozy- C WT rep E Posterior Pronephric Duct Blotted w/ gous carriers of hi2394. From three repeated experiments, an Rep IP -Rep average of 13.0% body curvature and 10.3% kidney cyst phe- notypes were observed in the reptin-egfp–injected group, com- Lysate -Tub pared with an average of 31.5% body curvature and 28.6% D kidney cyst phenotypes in the control group (Fig. 1D). Com- 40% bined, the above results demonstrate that hi2394 is a loss-of- 30% WT aTub Cdh17 DAPI rep aTub Cdh17 DAPI function allele of reptin. 20% * F Olfactory Placode G Lateral Line 10% Reptin ’ percentage of percentage Transcripts Are Enriched in Tissues with Motile Cilia. Reptin s body curvature body 0% gfp rep function in vertebrate development, aside from its potential role 40% 30% in heart growth (36), has not been examined. To understand the 20% role of reptin in development, we examined the expression pat- * 10% tern of reptin through multiple developmental stages via in situ kidney cyst kidney percentage of percentage 0% WT aTub rep aTub WT aTub F-act rep aTub F-act gfp rep hybridization. Reptin RNA was present as early as one- to two- cell stage in wild-type embryos (Fig. S2A). Because in zebrafish, Fig. 1. Phenotypes of reptinhi2394 mutants. (A) Reptinhi2394 mutant (rep)at zygotic transcription does not commence until around the 500– 3 d postfertilization (dpf) showing kidney cyst (red box, magnified in Lower 1,000-cell stage, this result indicates that reptin transcripts are fi Right) and body curvature, compared with wild-type (WT) sh. (B) RT-PCR deposited maternally. Up to the bud stage, reptin was expressed using lysates of 2-dpf reptinhi2394 (rep) and WT siblings. Two pairs of primers, one to the 5′ side of the proviral insertion and one to the 3′ side, were used. almost ubiquitously (Fig. S2A). Later, although still widely dis- (C) The greatly reduced Reptin protein in 4-dpf reptinhi2394 mutant (rep). tributed, reptin transcripts became more concentrated in highly (Upper) Samples precipitated and blotted with anti-Reptin. (Lower) Lysates ciliated tissues, including the neural tube, the lateral line organ, blotted with anti–β-tubulin as a loading control. (D) Microinjection of reptin- and the pronephric duct (Fig. S2A). In contrast, the control eGFP mRNA (rep) reduced the percentages of body curvature (Upper) and reptin sense mRNA probe failed to detect any signal, verifying + − kidney cysts (Lower)inembryosfromreptinhi2394 / crosses, with eGFP mRNA the specificity of the antisense probe (Fig.
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