WDR31 is a novel ciliopathy protein displaying functional redundancy with GTPase-activating proteins ELMOD and RP2 in recruiting BBSome to cilium


 The term “ciliopathy” refers to a group of over 35 rare disorders characterized by defective cilia and many overlapping clinical features, such as hydrocephalus, cerebellar vermis hypoplasia, polydactyly, and retinopathy. Even though many genes have been implicated in ciliopathies, the genetic pathogenesis in certain cases remains still undisclosed. Here, we identified a homozygous truncating variant in WDR31 in a patient with a typical ciliopathy phenotype encompassing congenital hydrocephalus, polydactyly, and renal agenesis. WDR31 is an evolutionarily conserved protein that localizes to the cilium and cilia-related compartment. Analysis from zebrafish supports the role of WDR31 in regulating the cilia morphology. The CRISPR/Cas9 knock-in (p.Arg261del) C. elegans model of the patient variant (p.Arg268*) reproduced several cilia-related defects observed in wdr-31 null mutants. Mechanistic analysis from C. elegans revealed that WDR-31 functions redundantly with ELDM-1 (ELMOD protein) and RPI-2 (RP2) to regulate the IFT trafficking through controlling the cilia entry of the BBSome. This work revealed WDR31 as a new ciliopathy protein that regulates IFT and BBSome trafficking.


Introduction
formation in all examined organisms, indicating the importance of IFT for cilia assembly (Pazour 91 et al., 2000;Prevo et al., 2017). 92 Bardet-Biedl syndrome (BBS) was classified as a ciliopathy in 2003, and eight of the highly 93 conserved Bardet-Biedl syndrome proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9, and 94 BBIP10) establish a stable protein complex called the BBSome that undergoes IFT. Work from a 95 range of organisms implicates the BBSome in a variety of cilia related process including acting as 96 a cargo adaptor for removing proteins from cilia and as a regulator of the assembly and stability 97 of IFT trains (Ansley et al., 2003;Lechtreck et al., 2009;Loktev et al., 2008;Nachury et al., 2007;98 Nozaki et al., 2019;Ou et al., 2007Ou et al., , 2005Wei et al., 2012;Williams et al., 2014;Xu et al., 2015; 99 Ye et al., 2018). For example, in the nematode C. elegans mutants lacking bbs-7 or bbs-8, 100 detachment of IFT-A and IFT-B in the amphid cilia was reported in the anterograde direction (Ou 101 et al., 2005). Analysis with a hypomorphic mutant (bbs-1) revealed that BBSome is involved in 102 attaching IFT-B components to the retrograde IFT machinery at the ciliary tips in the channel cilia 103 of C. elegans (Wei et al., 2012). 104 To identify new ciliopathy genes, we focused on our single-cell RNA-seq data that compared 105 expression profiles of ciliated cells with those of non-ciliated cells in the nematode C. elegans, and 106 this work revealed novel cilia genes, including WDR31 (manuscript in preparation). Our gene 107 discovery approach in combination with the use of clinical genetic testing and experimental 108 validations identified WDR31 (WD repeat domain 31) as a novel ciliopathy gene encoding a highly 109 conserved cilia-associated protein. Our work from Zebrafish and C. elegans provides significant 110 insight into the function of WDR31 in cilia biogenesis. First, our work from C. elegans revealed 111 that WDR-31 functions redundantly with two GTPase activating proteins (GAPs) ELMD-1 (the 112 sole ortholog of the human ELMOD proteins) and RPI-2 (human retinitis pigmentosa 2 113 orthologue) to control the cilia morphology. Second, knocking out wdr-31 along with elmd-1 or 114 elmd-1;rpi-2 causes IFT trafficking to be disrupted, resulting in ciliary tip accumulations of  B components and the OSM-3/KIF17 motor, as well as significantly reduced BBSome recruitment 116 to cilia. Taken together, WDR31 is a new ciliopathy-associated protein that exhibits functional 117 redundancy with two GAP proteins (ELMOD and RP2) in regulating cilia morphology and IFT 118 trafficking.

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C. elegans strains, maintenance and genetic crossing 122 For strain maintenance and genetic crosses, a standard procedure was followed, as described by 123 Sidney Brenner in 1974(Brenner, 1974. After genetic cross with a marker to generate single, 124 double and triple mutants, we used the PCR strategy to trace the mutations in following mutants:  127 Primers can be found in Table S1.

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N2;turEx21 [arl-13p::wdr-31 (T05A8.5 an empty sgRNA vector pRB1017. The successful sgRNA insert was confirmed with colony PCR, plasmid pRF4) (Dickinson et al., 2013). F1s with the roller phenotype were identified, and after 160 they generated enough progenies, the PCR technique was used to identify the F1 generation 161 bearing the predicted deletion, as well as the homozygosity of the allele mutation. The PCR 162 products from knockout animals were then sent to the Sanger sequencing (Macrogen, South 163 Korea). wdr-31(tur003)II. mutants have 1276-bp deletion covering a huge part of exon II (297 bp 164 out of 359 bp) and whole exon III, IV, and exon V. This is likely a null allele of wdr-31. sgRNA 165 sequences can be found in Table S1. with an Andor iXon Ultra 897 EMCCD (an electron-multiplying charge-coupled device camera).

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The LSM900 confocal microscope with Airyscan 2 (ZEN 3 Blue edition software) was used to 224 capture the high-resolution Z-stack images. On microscope slides, a drop of 2-3% agarose was 225 used to create an agarose pad, and worms were then moved to the agarose pad containing 1-3 μl 226 of 10 mM levamisole (an anesthetic agent). Images were collected at 0.14 m intervals with a Plan 227 ApoChromat 63x/1.40 NA target, then analyzed with Blue edition software ZEN 3 to create Z-228 stacks, and processed with ImageJ (NIH) software (Schneider et al., 2012).

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Osmotic Avoidance Assay for assessing cilia functions in C. elegans 239 Right before each experiment, we freshly prepared an 8M fructose solution containing 240 bromophenol blue and made a ring of 8M bromophenol blue solution using 1 ml pipette tips. 1-241 day adult worms of wild type and osm-5(p813) mutants were always tested as positive and negative 242 controls before evaluating the mutant strains (Haycraft et al., 2001). Wild-type worms are 243 supposed to remain in the ring under normal conditions, while osm-5(p813) mutants may be able 244 to escape due to a defect in the cilia structure. Every assay lasted 5 minutes, and five worms were 245 placed in the ring. At least three separate experiments (on different days) were conducted for each 246 strain, and the total number of worms was more than 40 for wild type and osm-5(p813), and more 247 than 80 for the rest of mutants. Fig. S4A was a combined representation of the last two independent 248 experiments.

Whole-mount in situ hybridization in zebrafish 251
Whole-mount in situ hybridization was performed as previously described (Thisse et al., 2004).

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The wdr31  Generation of zebrafish mutants using the CRISPR/Cas9 system was carried out as previously 262 described (Chang et al., 2013). Briefly, two gRNAs targeting sequences in wdr31 were chosen as into the embryos at one-cell stage.

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Sanger sequencing and segregation analysis were done for the proband and family members.

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Fisher's exact test). 435 We then sought to examine AWB cilia morphology in triple mutants and compared it with the would be unable to return from the ciliary tips due to potential defects in the IFT returning, so we 482 measured IFT particle frequency in wild type and affected mutants to explore if the number of IFT 483 particles entering into cilia is affected and/or if the return of IFT particles from the ciliary tips is 484 altered. Using time-lapse video coupled with kymography, for a subset of IFT components, we 485 discovered a substantial reduction in the number of IFT particles traveling along the cilia in both 486 directions in double and triple mutants (Fig. 6A, and B) and 11 in triple mutants; p < 0.0001; the Mann-Whitney U test) (Fig. 6B). Furthermore, we found The ciliopathy associated variant in WDR-31 displays cilia defects in C. elegans 508 Like human WDR31, C. elegans WDR-31 has WD-repeat domains (Fig. 3A, and Fig. S2A). In 509 the ciliopathy patient, we found the arginine-to-a termination codon substitution at residue 268 of 510 human WDR31 that removes the 99 amino acids containing a WD domain, but the functional 511 significance of this change is currently unknown in the context of cilia biogenesis. To investigate 512 whether the last 99 amino acids in WDR-31 is essential for the subcellular localization of WDR-513 31 to the cilia base and the function of WDR-31, the CRISPR/Cas9 was used to engineer the C. 514 elegans model harboring the corresponding WDR31 deletion (named WDR-31 R261-del ) (Fig. 7A).

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Finally, because WDR-31, ELMD-1, and RPI-2 localize at the base of cilia, we wanted to examine 529 the role of these proteins in the ciliary gate, and we chose the TRAM-1 protein (the ortholog of 530 human TRAM1), which surrounds the PCMC in C. elegans but stays outside of cilia (Williams et 531 al., 2011). Our confocal microscopy analysis revealed that the TRAM-1 protein enters into cilia in 532 all examined wdr-31 triple mutants, including wdr-31 R261-del ;elmd-1;rpi-2 but not in wdr-31 R261-del 533 single mutants (Fig. 7E). Taken together, all these data from C. elegans indicates the human R268* double and triple mutants relative to wild type (Fig. 8C). Our analysis revealed a substantial 552 decrease in the average of BBS-7::GFP particles translocating in the anterograde and retrograde 553 directions in the remaining worms that display IFT (average anterograde and retrograde IFT 554 particles: 18 and 19 for wild type; 10 and 11 for double mutant; 2 and 2 for triple mutant, p < 555 0.0001; the Mann-Whitney U test) (Fig. 8C, D and Supplementary Movie S6). We next explored 556 the localization of transition fiber protein FBF-1 and TAX-4 (a membrane protein) in the triple 557 mutants because FBF-1 was implicated in the ciliary entry of IFT machinery (Wei et al., 2013).

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Our data reveals that the localization of these proteins remains unaffected in the triple mutants 559 ( Fig. 5SB and D). Taken together, our results suggest that because the BBSome complex is unable  containing protein in Penicillium camemberti (Gabler et al., 2020). PSI-BLAST search confirmed 625 this result (unpublished data).

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Furthermore, the ciliary roles of these two GAP proteins might be independent of their GAP 628 activities, and consistent with this idea, the recent study showed the ciliary roles of ELMOD2 are 629 partially independent of its GAP activity (Turn et al., 2021). However, a variety of evidence                 Confocal images show colocalization of TAX-4 (a ciliary membrane protein) and XBX-871 1::tdTomato (a cilia marker) or NPHP-1::GFP (transition zone protein) and FBF-1:: mCherry 872 (transition fiber protein) or PLCδ1-PH::GFP (a marker for monitoring phosphatidylinositol 4,5-873 bisphosphate (PtdIns(4,5)P2) in the plasma membrane) and MKSR-2 (a TZ marker). The  were processed with Image J to generate GIFs (10 fps).