Biochimica et Biophysica Acta 1863 (2016) 1307–1318

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Biochimica et Biophysica Acta

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Genome-wide screen identifies novel machineries required for both ciliogenesis and arrest upon serum starvation

Ji Hyun Kim a,1, Soo Mi Ki a,1,Je-GunJoungb, Eric Scott c, Susanne Heynen-Genel d, Pedro Aza-Blanc d, ChangHyukKwonb,JoonKime,JosephG.Gleesonc,⁎, Ji Eun Lee a,b,⁎⁎ a Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, #81 Irwon-Ro Gangnam-Gu, Seoul 06351, Republic of Korea b SGI, Samsung Medical Center, #81 Irwon-Ro Gangnam-Gu, Seoul 06351, Republic of Korea c Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA d High Content Screening and Functional Genomics Core, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA e GSMSE, KAIST, 291 Daehak-Ro, Yuseong-gu, Daejeon 34141, Republic of Korea article info abstract

Article history: Biogenesis of the primary cilium, a cellular organelle mediating various signaling pathways, is generally coordi- Received 12 October 2015 nated with cell cycle exit/re-entry. Although the dynamic cell cycle-associated profile of the primary cilium has Received in revised form 19 March 2016 been largely accepted, the mechanism governing the link between ciliogenesis and cell cycle progression has Accepted 22 March 2016 been poorly understood. Using a -wide RNAi screen, we identify encoding subunits of Available online 28 March 2016 the spliceosome and as novel regulators of ciliogenesis. We demonstrate that 1) the mRNA Keywords: processing-related hits are essential for RNA expression of molecules acting in cilia disassembly, such as – High-content screen AURKA and PLK1, and 2) the proteasome systems (UPS)-involved hits are necessary for Ciliogenesis of molecules acting in cilia assembly, such as IFT88 and CPAP. In particular, we show that these screen Cell cycle hit-associated mechanisms are crucial for both cilia assembly and cell cycle arrest in response to serum with- mRNA processing drawal. Finally, our data suggest that the mRNA processing mechanism may modulate the UPS-dependent Ubiquitin–proteasome system decay of cilia assembly regulators to control ciliary resorption-coupled cell cycle re-entry. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction axonemal microtubules [5]. Because of the ambilateral roles of the mother centriole in ciliogenesis and cell division, the events of cilia bio- For many years, the primary cilium had been considered an obscure genesis and cell cycle progression are mutually exclusive. structure of no importance. However, recent studies suggest that this In general, assembly of the primary cilium is coupled with cell cycle cellular organelle acts as a major hub for multiple signal transduction exit and entry into quiescence, whereas its disassembly is coupled with pathways that are necessary for diverse cellular phenomena, including resumption of proliferation [6–8]; these facts present long-standing differentiation, polarity, and homeostasis [1–3]. The primary cilium is evidence for a link between ciliogenesis and cell cycle progression. an immotile and microtubule-based sensory organelle that extends Although the idea that cilia are resorbed as the cells enter mitosis was from a mother centriole in most types of cells. The mother centriole gen- once prevalent, recent studies have indicated that cilia disassembly pro- erally contributes to formation of the mitotic spindle in a dividing cell, ceeds in two waves as quiescent cells re-enter the cell cycle: the first but in a quiescent cell it becomes associated with a Golgi-derived vesi- wave occurs at the G1 → S transition, and the second wave occurs at cle, migrates to the cell surface, and attaches to the plasma membrane the G2 → Mtransition[9]. It has also been suggested that the inhibition [4]. Once it attaches, the mother centriole is referred to as the basal of ciliary resorption halts cell cycle progression. For example, depletion body, which serves as a nucleation region for the growth of ciliary of Nde1, Tctex-1, and Aurora kinase A (AURKA) results in the delay of cell cycle re-entry with abnormal retention of cilia [10,11]. Thus, it ap- pears that not only is a suppressive mechanism of ciliogenesis active ⁎ Corresponding author. during cell proliferation, but a stimulatory mechanism of ciliary resorp- ⁎⁎ Correspondence to: Ji Eun Lee, Department of Health Sciences and Technology, tion may also be active. Together, these observations imply that the dis- SAIHST, Sungkyunkwan University, #81 Irwon-Ro Gangnam-Gu, Seoul 06351, Republic assembly of the primary cilium is a prerequisite for cells to cease being of Korea. quiescent and re-enter the cell cycle. E-mail addresses: [email protected] (J.G. Gleeson), [email protected] – (J.E. Lee). Recent studies have shown that autophagy- and ubiquitin pro- 1 These authors contributed equally to this work. teasome system (UPS)-mediated proteolysis of cilia-related

http://dx.doi.org/10.1016/j.bbamcr.2016.03.021 0167-4889/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1308 J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318 molecules are involved in cell cycle exit and subsequent cilia assem- targeted each (Supplementary Table S1). The cells were then sub- bly [12,13]. For instance, UPS-mediated degradation of Trichoplein, jected to automated fluorescent imaging and scoring followed by visual which is composed of Cul3-RING ubiquitin ligases (CRL3s) and inspection (Fig. 1E) and secondary screening in triplicate. Through man- KCTD17 as a substrate-adaptor, inactivates AURKA and initiates ual visual inspection, we confirmed that the non-specificscrambled ciliogenesis [13]. Therefore, it is conceivable that the formation of siRNA-transfected cells under serum starvation had mostly EGFP- the primary cilium is regulated by degradation of involved labeled cilia and few cells with mCherry-labeled nuclei. Finally, we ob- in cilia disassembly prior to exit from the cell cycle. Although there tained the screen hits, which displayed abnormal numbers of EGFP- are several clues as to the basis of coordinated regulation of cilia and mCherry-nuclei with Z-scores N2; the hits were clustered ciliogenesis and cell cycle progression, some questions remain into three outcome groups (Fig. 1F). Among the classified hits that about how resorption of the primary cilium modulates progression surpassed the threshold, we identified 201 in Group A, 460 in Group of the cell cycle. A mechanistic explanation does not exist for how B, and 164 in Group C (Supplementary Table S2). In addition, we thepresenceorabsenceoftheprimaryciliumcanserveasacheck- found that several genes such as NEK2, PLK1, APCs, and cyclins, which point at the G1 → Stransition. are known to be associated with ciliogenesis-linked cell cycle control, In the present study, we perform a genome-wide high-content were included in our screen hits. screen and uncover roles of mRNA processing and UPS mechanisms in Because the hits from Groups A, B, and C were likely to be largely cell cycle arrest and ciliogenesis. We find that mRNA processing- non-overlapping, we profiledtheresultsseparately.GroupAhada related hits are required to control RNA expression of cilia disassembly normal number of EGFP-cilia but a higher than normal number of regulators (such as AURKA, PLK1, and NEK2), and UPS-related hits are mCherry-nuclei. This result indicated that silencing of Group A necessary for the proteolysis of cilia assembly regulators (such as genes might cause inhibition of ciliary resorption and failed entry CPAP, KATANIN, and IFT88). Furthermore, our data suggest that mRNA into mitosis. Group B had a lower than normal number of EGFP- processing is a stimulatory mechanism of cilia disassembly, whereas cilia but a normal number of mCherry-nuclei, suggesting that the UPS is a suppressive mechanism of cilia assembly. Finally, we demon- Group B genes might have no direct effect on the cell cycle but strate that the mRNA processing and UPS-mediated ciliary resorption might block ciliogenesis. Furthermore, we found that the Group B provide a link to the G1 → S transition during cell cycle re-entry. list included many known ciliopathy genes and, accordingly, identi- fied a novel Joubert Syndrome causative gene, KIAA0586,fromthe 2. Results list [14]. Group C had a lower than normal number of EGFP-cilia and a higher than normal number of mCherry-nuclei. This finding 2.1. Identification of novel modulators of ciliogenesis and cell cycle suggested that depletion of Group C genes triggered a blockade of progression entry into or maintenance of a quiescent state and a concurrent (or resulting) failure of ciliation. Therefore, we focused on Group C be- To obtain a list of candidates and to gain a comprehensive under- cause the results suggested dual regulation of ciliogenesis and cell standing of the molecular mechanism connecting ciliogenesis to cell cycle progression. To extract information regarding the mechanisms cycle progression, we utilized a functional genomics approach. We per- by which ciliogenesis is coupled to cell cycle regulation, we analyzed formed a high-content screening (HCS) procedure using small interfer- the list of hits using public databases such as DAVID [17] and ing RNA (siRNA) [14] to analyze the genetic intersection of ciliogenesis GeneMANIA [18]. The results of combined analyses showed enrich- and cell cycle state (Fig. 1A). This screen utilized a subclone of the ment within two functional clusters: mRNA processing and the ubiq- hTERT-immortalized ciliary retinal pigment epithelial (RPE) cell line ex- uitin–proteasome system (UPS) (Fig. 1G). The mRNA processing pressing both EGFP-tagged Smoothened, a transmembrane that module (GO: 0006397) is involved in the conversion of a primary accumulates in the cilium [15], and the N-terminal 110 amino acid res- mRNA transcript into one or more mature mRNAs prior to transla- idues of Geminin, a nuclear protein stabilized during S, G2, and M tion into a polypeptide. The UPS module (proteasome-mediated phases, fused to mCherry [16] (Fig. 1B). We predicted that this cell ubiquitin-dependent protein catabolic process; GO: 0043161) is line, abbreviated SEMG (Smo-EGFP and mCherry-Geminin), would dis- involved in the breakdown of a protein (this process is initiated by play a green fluorescent primary cilium during the G0/G1 phases and a attachment of ubiquitin, which is mediated by the proteasome). red fluorescent nucleus during S, G2, and M phases (Fig. 1C). Because serum starvation induces cell cycle arrest in the G0/G1 2.2. The machineries of mRNA processing and UPS are necessary for phases and ciliary assembly in RPE cells, we hypothesized that serum ciliogenesis and cell cycle control withdrawal would yield a majority of cells with EGFP-positive cilia and mCherry-negative nuclei, whereas addition of serum would cause To validate the role of hit genes in the coupling of ciliogenesis the majority of the cells to display mCherry-positive nuclei with EGFP- with cell cycle progression, we performed fluorescent imaging negative cilia. The cells were subjected to automated fluorescent imag- and fluorescence-activated cell sorting (FACS) analyses on knock- ing and scoring, and we found that ~70% of SEMG cells that were down cells transfected with individual siRNAs against representa- cultured in the presence of fetal bovine serum (FBS) showed tive hits. For the validation tests, we prioritized groups of genes mCherry-positive nuclei, with few if any EGFP-positive cilia. In contrast, that were predicted to have roles in similar pathways within each 24 h after serum deprivation, ~60% of the cells had EGFP-positive cilia cluster shown in Fig. 1G. The individual siRNAs used to target with few mCherry-positive nuclei (Fig. 1D). Additionally, we found each hit were selected based on a qRT-PCR knockdown efficiency that the depletion of positive control genes including KIF3A, ACTR3, test (Supplementary Fig. S1). We counted Smo-EGFP-positive and CRNKL1, which are involved in ciliogenesis or cell cycle regulation, ciliated cells and mCherry-Geminin-positive cycling cells from the im- led to abnormal numbers of EGFP-cilia and mCherry-nuclei in the aging data and compared the results to the FACS data. In the mRNA pro- serum-starved SEMG cells [14]. The rationale for performing our cessing pathway cluster, crooked neck pre-mRNA splicing factor 1 genome-wide RNAi screen with SEMG cells was to identify genes that, (CRNKL1); eukaryotic translation initiation factor 3, subunit E (EIF3E); when downregulated, interfered with the mutually exclusive expres- pre-mRNA-processing factor 6 (PRPF6); and pre-mRNA-processing fac- sion of mCherry and EGFP. tor 8 (PRPF8) were selected. As expected, the depletion of each hit A total of 18,055 target genes representing the known human resulted in a decrease of Smo-EGFP-positive cells and an increase of ENCODE transcriptome were individually knocked down in duplicate mCherry-Geminin-positive cells (Fig. 2A). Further analysis using FACS with the siRNA library for 72 h, including 24 h of serum starvation implied that the increased number of cycling cells might be due to an (Fig. 1A). The siRNA library was pooled such that four individual siRNAs increase of S phase cells (Fig. 2B). In the UPS pathway cluster, J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318 1309 AB Scheme of High-content siRNA screen Construct I: Cell cycle indicator S/G2/M: Red (Nucleus) mCherry Geminin (1-110) 1. Pooled siRNA transfection G0/G1: No expression (18,055 genes in duplicate) Construct II: Primary cilium marker G0/G1: Green (Primary cilium) 2. 72h incubation Smoothened (Smo) EGFP S/G2/M: Green (Cytosol) (24h serum starvation) C SEMG cell: hTERT-RPE1 cell expressing Smo-EGFP-mCherry-Geminin 3. Fixation and DAPI staining G0/G1 S G2 M Ciliated cells (Green +) Cycling cells (Red +) 4. High-content fluorescence imaging (Opera QEHS system) D E Nucleus Cytoplasm Normal Group A

5. Automated scoring (Acapella 2.0 program ) Z-score ranking>2.0 replicates Cilia (Serum +) Cilia (Serum -) Group B Group C 6. Visual inpection of images (CyteSeer 2.7 program)

7. Confirmation with individual siRNAs

FG Screen hit classification

Phenotype Group Ciliation Cell cycle

Normal Generation of cilia Quiescent state

A Generation of cilia Cycling state mRNA processing pathway B Reduction of cilia Quiescent state

C Reduction of cilia Cycling state

Ubiquitin-proteasome pathway

Fig. 1. High-content RNAi screen to identify novel regulators of ciliogenesis and cell cycle. A. The workflow started with reverse transfection of the pooled siRNA library in a medium for 72 h, with 24 h of these involving serum starvation. We then performed fixation, DAPI staining, fluorescent imaging, identification of hits based on Z-score ranking, and visual inspection of the hits. B. The mCherry-Geminin (1–110) construct was transfected as a cell cycle indicator and was expressed in the nucleus during the S, G2, and M phases of the cell cycle. The Smo-EGFP construct was transfected as a primary cilium marker and was expressed in the cytosol of cycling cells and primary cilia of quiescent cells. C. The hTERT-RPE1 cell line that was stably transfected with mCherry-Geminin (1–110) and Smo-EGFP was named the SEMG cell line. The cells showed prominent expression of EGFP in the primary cilium in the G0/ G1 phases and mCherry in the nucleus at S, G2, and M phases. D. The algorithm properly segmented the nucleus, cytoplasm, and primary cilium in SEMG cells under the conditions with and without serum. E. The screen hits were analyzed by manual image inspection using Cytseer 2.7 and classified into three groups. F. The classified hits were profiled in each group on the basis of the knockdown phenotype (red letters) in terms of ciliogenesis and cell cycle progression. G. The hit genes were clustered into two main functional groups, mRNA processing and UPS, by network analysis using GeneMANIA. Scale bars, 10 μm(E). proteasome subunit type-3 (PSMB3); proteasome 26S subunit, non- The FACS analysis also suggested that the increased number of cycling ATPase 8 (PSMD8); and proteasome 26S subunit, non-ATPase 11 cells was caused by an increase of S phase cells after gene silencing (PSMD11) were chosen; proteasome 26S subunit, non-ATPase 1 (Fig. 2D). We therefore focused on the common knockdown pheno- (PSMD1), which is not a hit from the screen, was also included. Because types of mRNA processing and UPS-related hits, which were a decrease 11 over 30 human proteasome subunits were hits in our screen (Fig. 1G of ciliated cells and an increase of S phase cells. and Supplementary Fig. S2), we wanted to determine if any specific It seemed that the increased number of S phase cells was correlat- subunit of the proteasome complex plays a role in the link between ed with the decreased number of G0/G1 phase cells (Fig. 2B, D). From ciliogenesis and cell cycle progression. The depletion of the chosen pro- the data, the cell cycle-related roles of the screen hits were inferred, teasome subunits showed decreases of Smo-EGFP-positive cells and in- and we predicted that the silencing of the hits might lead to the by- creases of mCherry-Geminin-positive cells that were consistent with pass of G0 arrest from G1 phase or a failure to maintain G0/G1 arrest the effects of knocking down mRNA processing-related hits (Fig. 2C). under serum starvation. We therefore hypothesized that the hit 1310 J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318

A ( Serum - ) mCherry-Geminin B ( Serum - ) G0/G1 50 S Smo-EGFP 100 G2/M 95 40 * 90 30 85 ** * 80 20 **

Cells (%) Cells ***

Cells (%) Cells 75 * * ** 70 10 15

5 0 (siRNAs) 0 (siRNAs) Ctrl EIF3E Ctrl PRPF6 PRPF8 EIF3E CRNKL1 PRPF6 PRPF8 CRNKL1

C ( Serum - ) mCherry-Geminin D ( Serum - ) G0/G1 60 S Smo-EGFP 100 G2/M 50 95 90 40 85 80 30 ** 75

Cells (%) Cells * 20 *** *** * (%) Cells ** 70 ** 15 10 *** 5 0 (siRNAs) 0 (siRNAs)

Ctrl Ctrl PSMB3 PSMD1 PSMD8 PSMB3 PSMD1 PSMD8 PSMD11 PSMD11 mCherry-Geminin E Smo-EGFP 50 ( Serum + ) G *** *** 40 *** Serum - (FBS 0%) *** ** M M 30 G2 G2G ** G1 G0 20 G1 G0

Cells (%) Cells ** * * S S * 10 ** ** ** ** ** *** Ctrl siRNA Hit siRNA 0 (siRNAs) Assembly of cilia - A decrease of ciliated cells Ctrl EIF3E in quiescent cells - An increase of S phase cells PRPF6 PRPF8 PSMB3 PSMD1 CRNKL1 PSMD8 PSMD11

F ( Serum + ) G0/G1 Serum + (FBS 10%) 100 S 95 G2/M M M 90 G2G G2

85 G1 G0 G1 G0 80 S S

75 Cells (%) Cells 70 Ctrl siRNA Hit siRNA 15 5 Disassembly of cilia - An increase of ciliated cells 0 (siRNAs) in cycling cells - A decrease of S phase cells

Ctrl EIF3E PRPF6 PRPF8 PSMB3 PSMD1 PSMD8 CRNKL1 PSMD11

Fig. 2. Validation of hit genes involved in the coupling of ciliogenesis with cell cycle control. A. The SEMG cells were transfected with individual siRNAs targeting mRNA processing-related hits under serum starvation and subjected to fluorescent imaging to analyze ciliation (Smo-EGFP) and cell cycling (mCherry-Geminin). B. The cell cycle state of the hit-silenced SEMG cells was analyzed by FACS under serum starvation. C. The SEMG cells were transfected with individual siRNAs targeting proteasome subunits under serum starvation and subjected to fluorescent imaging to analyze ciliation and cell cycling. D. The cell cycle state of the proteasome subunit-silenced SEMG cells was analyzed by FACS under serum starvation. E. The screen hit-depleted SEMG cells were subjected to fluorescent imaging to analyze ciliation and cell cycling in the presence of serum. F. The cell cycle state of the hit gene-depleted SEMG cells was analyzed by FACS in the presence of serum. The data are shown as the mean ± SEM, One-way ANOVA: *P b 0.05, **P b 0.005, and ***P b 0.001 (n = 3, [A, C, and E]). G. Based on the results of fluorescent imaging and FACS analyses, the roles of screening hit genes were predicted. genes played roles in G1 phase and that the dysregulation of hit- showed that the silencing of hits caused an increase in the number mediated mechanisms resulted in the abnormal transition of cells of G0/G1 arrested cells (Fig. 2F). These data implied possible roles to S phase from G1 phase. To determine if function of the screen for the screen hits in the regulation of the G1 → S transition as well hits during G1 phase affect the G1 → S transition, we simply exam- as ciliogenesis (Fig. 2G). Taken together, our validation analysis ined the down-regulation effect of the hits on cell cycle progression with the screen hits showed not only that our screening was robust in the presence of serum (Fig. 2E, F, and Supplementary Fig. S3A). but also that mRNA processing- and UPS-associated mechanisms The knockdown led to more Smo-EGFP-positive cells and fewer might be important for controlling coordination of the G1 → Stran- mCherry-Geminin-positive cells (Fig. 2E). Remarkably, FACS data sition of the cell cycle with cilia biogenesis. J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318 1311

2.3. The mRNA processing and UPS mechanisms are essential for ciliary zebrafish (Supplementary Fig. S3B). We found that the zebrafish larvae formation and function in vivo treated with SSA or MG132 and injected with prpf8 MOs or MOs showed typical ciliary defects, such as peripheral heart edema, small Because most of the screen hits have not been previously reported as brain/hydrocephalus, abnormal otoliths (abnormal angle between two ciliary genes, we wondered whether the machineries for mRNA pro- otholiths) and curved tails [20] (Fig. 3A, B, and Supplementary cessing and UPS are necessary for ciliogenesis/ciliary function in vivo. Fig. S4A). According to previous findings showing that malformed or We therefore examined the inhibitory effect of mRNA splicing by utiliz- dysfunctional cilia result in disruption of heart asymmetry zebrafish ing spliceostatin A (SSA) [19] and morpholinos (MOs) targeting prpf8, [20,21], we analyzed the heart laterality of the embryos of Tg(flk1:egfp) and that of UPS by utilizing MG132 and MOs targeting psmd8 in zebrafish (an endocardium-specific transgenic reporter line) treated

A B Severe Mild DMSO 1% SSA 40 µM MG132 50 µM Normal * 100 80 n=24 n=56 n=17

A P 72hpf 60 n=61

Control MO prpf8 MO psmd8 MO 40 n=80 n=28 n=22 n=51 * * (%) Embryos 20 n=14 0 Control prpf8 MO SSA psmd8 MO MG132 A P 72hpf (n=81) (n=84) (n=66) (n=111) (n=48)

C E Abnormal Control prpf8 MO SSA Normal 100

V V 80 V A A A A 60 n=93 n=85 n=42 P flk1:EGFP 40

D Embryos (%) 20 Normal Midline 0 Control prpf8 MO SSA (n=100) (n=104) (n=50) 48hpf 48hpf

A V F V olf. olf. LR A A P

G A LR 72hpf DMSO DMSO Control MO Control MO P

A H Abnormal GT335 GT335 P Normal 100 SSA SSA prpf8 MO prpf8 MO 80

60 n=72 n=36 n=52 n=31 n=39 GT335 GT335 40 MG132 MG132 psmd8 MO psmd8 MO

Embryos (%) 20

0 Control prpf8 SSA psmd8 MG132 GT335 GT335 (n=73) (n=51) (n=66) (n=51) (n=48)

Fig. 3. The function of spliceosome and proteasome is essential for ciliogenesis in vivo. A. Overall morphological defects in the MOs-injected, SSA- and MG132-treated zebrafish embryos at 72 hpf. Arrows indicate heart edema; arrowheads point hydrocephalus; asterisks indicate otolith defects in the ear. B. The embryos showing ciliary defects are quantified according to severity, and the data were presented in the graph. C. The asymmetry of the heart, controlled by cilia in Kupffer's vesicle, was observed in the prpf8 MO-injected and SSA-treated Tg(flk1:EGFP) zebrafish larvae. V: ventricle, A: atrium. D. The diagrams show normal and abnormal (midline) heart asymmetry of the zebrafish at 48 hpf. E. The Tg(flk1:EGFP)zebrafish larvae showing defects in heart asymmetry were counted and the quantified data were presented in the graph. F. The diagram shows head area of the zebrafish larva containing olfactory organs at 72 hpf. The rectangle indicates the photographed region after immunostaining with anti-GT335 antibodies. Olf.: olfactory organs, hpf: hours post-fertilization. G. Zebrafish larvae were treated with 40 μM SSA and 50 μM MG132 to inhibit function of the spliceosome and proteasome, respectively. The larvae were immunostained with anti- GT335 antibodies and the olfactory regions were compared to those of the DMSO-treated control larvae at 72 hpf. The olfactory cilia of zebrafish larvae, injected with control MO, prpf8 MO, or psdm8 MO were also observed by GT335 immunostaining. The magnified images are indicated by insets and presented in the right panels. H. The individual zebrafish larvae showing ciliogenesis defects within the olfactory organs were counted. The quantified data were presented in the graph. A: anterior, P: posterior, L: left, R: right (A, C, D, F, and G). 1312 J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318 with SSA or MG132 and injected with prpf8 or psmd8 MOs. We found four candidates in the knockdown cells were lower than those of control that the inhibition of the spliceosome by SSA and prpf8 MOs caused cells (Fig. 4D). Taken together, these data suggest an influence of the failed laterality of the ventricle and atrium in the zebrafish heart mRNA processing mechanism on the cilia resorption and G1 → S transi- (Fig. 3C–E). Moreover, the drug-treated or MOs-injected larvae showed tion mediated by ciliary genes, such as AURKA, PLK1, NEK2,andCCNB1 attenuated ciliary formation in the cells of the olfactory organ at 72 h (Fig. 4E). post-fertilization (hpf) (Fig. 3F–H). We tested the rescue for psmd8 MO, but not for prpf8 MO, because the coding sequence of zebrafish 2.5. Proteolysis of cilia assembly regulators promotes ciliary resorption and prpf8 was very large to obtain an expression construct. We found that cell cycle re-entry the morphological defects were not due to off-target effects of MOs (Supplementary Fig. S4B–C) and also confirmed that the reduced cilia Next, to elucidate a required role of the UPS mechanism in the link were not due to less olfactory cells by drug-treatment or MO injection between cilia disassembly and cell cycle re-entry, we additionally com- (Supplementary Fig. S5). Taken together, these data suggest essential pared the phenotypes of SEMG cells treated with MG132 to those of roles of mRNA processing, such as RNA splicing, and the UPS in the reg- proteasome subunit-depleted cells. Both the knockdown and MG132- ulation of ciliogenesis and ciliary function in vivo. treated cells displayed an inhibition of ciliary resorption and cell cycle re-entry 18 h after serum re-addition (Fig. 5A and Supplementary 2.4. The control of cilia disassembly regulator expression is crucial for the Fig. S3C). Moreover, the disruption of the UPS mechanism led to an in- link between ciliary resorption and cell cycle re-entry creased number of cells in the G0/G1 phases, indicating an important role of UPS in cilia disassembly and control the G1 → S transition Several studies have shown that most cells with cilia in the G0/G1 (Fig. 5B). arrested state lose them when the quiescent cells re-enter the cell Because the function of the proteasome complex has not been previ- cycle [8,22]. According to our validation data, suggestive of a ciliary ously implicated in ciliary resorption, we assumed that the cilia- disassembly-associated role by the molecular mechanism acting in the associated molecules undergoing UPS-dependent proteolysis might be G1 → S transition, we assessed the knockdown effects of the screen key regulators that connect cell cycle re-entry to cilia disassembly. hits on cilia disassembly. We first explored the mRNA processing regu- Based on previous studies, we selected several ciliary regulators as can- lators including CRNKL1, EIF3E, PRPF6,andPRPF8 (Fig. 3). In general, the didates [28,30–34]. We then examined the protein expression levels of human telomerase reverse transcriptase-transformed retinal pigment the candidate molecules by Western blot analysis. We found that the epithelium (hTERT-RPE1) cells that are forced to generate cilia by protein levels of cilia assembly regulators, such as CPAP, KATANIN, serum deprivation and resorb the cilia 18 h after serum is added [22]. and IFT88, were reduced in control cells 18 h after serum addition, The cell cycle states of SEMG cells were also changed by incubation whereas the protein levels in the knockdown and MG132-treated cells with serum (Supplementary Fig. S3C, S6). In line with other studies, were as high as or higher than those of control cells under serum depri- control siRNA-transfected SEMG cells were forced to disassemble cilia vation (Fig. 5C, D). and re-enter the cell cycle 18 h after cessation of serum starvation. In It has been reported that the hTERT-RPE1 cells undergo cilia disas- contrast, knockdown of the mRNA processing-related hits blocked sembly at two different time points, in early G1 and S phases, 2 and cilia disassembly and cell cycle re-entry in SEMG cells (Fig. 4A). Accord- 18 h after serum re-addition, respectively [22,35].Therefore,inG1 ing to FACS analysis, hit gene-silenced SEMG cells maintained G0/G1 phase we analyzed the protein levels of HEF1 and MPS1, which were be- arrest instead of re-entering the cell cycle after addition of serum lieved to be important for the G0 → G1 transition and cilia disassembly (Fig. 4B). These results indicated that the mRNA processing mechanism [36–38] (Supplementary Fig. S3D). Consistent with other studies, serum is important for ciliary resorption and the G1 → Stransitionincellcycle addition induced higher protein expression of HEF1 and MPS1 than con- re-entry. trols under serum deprivation. However, the serum-induced increase of To determine the possible molecular mechanism, we performed HEF1 and MPS1 protein levels in the UPS-dysregulated cells was greater whole transcriptome sequencing (RNA-Seq), which can analyze overall than that of controls (Fig. 5E, F). In S phase, we analyzed the protein mRNA expression and pre-mRNA splicing events. We compared the levels of AURKA and PLK1, which were known to play a role in cilia dis- RNA-Seq results of CRNKL1-, EIF3E-, PRPF6-, and PRPF8-depleted cells assembly and be highly expressed 18 h after serum addition in the RPE1 with those of control siRNA knockdown cells. The results showed that cells [39]. We found that their protein levels in the UPS-deregulated few genes caused alternative splicing, and the genes were not common cells were not appreciably different from those of controls, which among the knockdown cells. However, expression levels of several were increased by serum re-addition (Supplementary Fig. S7A, B). genes were changed and commonly targeted (Fig. 4C and Supplementa- Taken together, our data suggest that the UPS-dependent decay of the ry Table S3). This result was not surprising because the mRNA process- ciliary proteins is necessary for cilia resorption and cell cycle re-entry ing hits included not only splicing factors but also translation regulators in G1 phase. Finally, these results imply a dual role of UPS in G1 such as EIF3E, which controls mRNA expression of target genes by reg- phase: 1) Proteolysis of any suppressor of cilia disassembly regulators ulating nonsense-mediated mRNA decay [23–25]. Furthermore, it has such as HEF1 and MPS1 to control G0 → G1 transition; and 2) Proteolysis been reported that splicing factors also regulate expression of mRNA of cilia assembly regulators to control G1 → S transitions. via direct interaction with mature RNA [26]. We hypothesized that the knockdown phenotype would involve altered expression of certain 2.6. The mechanisms to control expression of ciliary molecules regulate the genes crucial for either ciliogenesis or cell cycle control. Accordingly, cell cycle progression the heatmap generated by analysis of the genes commonly targeted by the screen hits showed an obvious contrast between the clusters of The similar effects of mRNA processing- and UPS-dysregulation on genes with decreased and increased expression levels (Fig. 4C). Within ciliogenesis and cell cycle progression prompted us to explore a func- the cluster of genes with decreased expression were AURKA, PLK1, NEK2, tional relationship between these two mechanisms. Although the func- and CCNB1. These genes have been reported to play a role in cilia disas- tional importance of the mRNA processing mechanism was in the sembly, which is independent of their well-known roles in mitotic regulation of expression of AURKA and PLK1, the protein levels were control of the cell cycle [22,27–29]. Moreover, our data were consistent not affected by UPS deficiency 18 h after serum re-addition (Fig. 4C with those of another report showing that knockdown of AURKA gave and Supplementary Fig. S7A, B). Furthermore, 2 h after cessation of rise to G0/G1 phase arrest and ablated cilia disassembly in hTERT- serum starvation, when AURKA and PLK1 were normally activated for RPE1 cells [30]. Upon validation with quantitative real-time PCR (qRT- cilia disassembly [37], no obvious change of their protein levels was PCR) analysis, we confirmed that the mRNA expression levels of all observed (Supplementary Fig. S7C, D). The results suggested that the J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318 1313

A mCherry-Geminin C 60 Smo-EGFP *** 50

40 CENPM CCNB1 CDC25B CCNB1 APOBEC3A,APOBEC3B

BIRC5

CDKN3

PBK

NUSAP1

CCNB2

UBE2T

NEK2

STMN1 30 CDKN2C NEK2 AURKA

ASF1B

SAPCD2

KIFC1 UCP2 AURKA C16orf59

RNASEH2A

HJURP Cells (%)

CDC20

CDCA8 20 AURKB SPC25

HINT2

BUB1

MYBL2

PRC1

TPX2

NDC80

CEP55

NUF2

DLGAP5

C15orf23 10 POC1A CDC25C

KIF2C

PIF1

OIP5

H2AFZ

CENPA

DBI

H2AFX

PADI1

MAD2L2

PSRC1

0 TNFAIP8L1

NRGN

TROAP

CKS2 Ctrl CRNKL1 EIF3E PRPF6 PRPF8 (siRNAs) UBE2S UBE2C

FAM83D

MPST

TUBB4B

HMGB2

FAM64A

PTTG1

PLK1 TK1 PLK1 PHGDH

PSAT1

ASNS

EIF4EBP1 Ctrl siRNA (-) Ctrl siRNA (+) PCK2 CHAC1

DTYMK

SGK196

HIPK2

ITGB8

VCAN

GPC6

FAT4

FNDC3B

ITGA11

BDKRB2

DPP4

KIAA1024

IL33

FAM198B

C1S

EGR1

CLSTN2

IL6ST

RRM2B

HEG1

NDRG4

LPHN2

SCG2

COLEC12

GPAM

ANGPTL4

NPTX1

KIAA1199

RSPO3

PRPF3

C1QTNF1

BTG2

KIAA0247

C3,TNFSF14

ICAM1

IL8

SERPINB2

PTGS2

IGFBP1

MMP14

CSF1

RRAD

CYP1B1

KCNJ2

ATP2B1

CACNA2D1

SYNPO2

BAMBI

LRIG3

BMF

ZFHX3

KIAA1644

ZMIZ1

DIO2

MMP1

ATP7A

C5orf41 CRNKL1 siRNA (+) EIF3E siRNA (+) PTPRU LRP1

SLC16A7

IGF2R

ODZ4 PRPF8 CRNKL1 PRPF6 EIF3E Ctrl

D 1.2 CCNB1 NEK2 1.0 PLK1 PRPF6 siRNA (+) PRPF8 siRNA (+) 0.8 AURKA

0.6

0.4

0.2 Relative RNA expression change) (fold 0 Ctrl PRPF6 EIF3E PRPF8 CRNKL1 (siRNAs)

B *** 100 Pre-mRNA G0/G1 E 95 AAAA... S 90 G2/M mRNA processing 85 regulators 80 mRNA AAAA... 75

Cells (%) 70 65 Cilia disassembly 60 regulators 20

0 (siRNAs) Ctrl G0/G1 S EIF3E PRPF6 PRPF8 CRNKL1

Fig. 4. The mRNA processing hits control RNA expression of cilia disassembly regulators and promote the G1–S transition. A. The ciliation and cell cycle of the mRNA processing hit-silenced SEMG cells were analyzed by fluorescent imaging analysis 18 h after cessation of serum starvation. The cells showed inhibition of cilia disassembly and cell cycle re-entry. (−), serum starvation; (+), serum re-addition for 18 h. B. The cell cycle state of the knockdown cells was analyzed by FACS and compared to that of controls after addition of serum for 18 h. The quantified data were presented in the graph. C. Whole-transcriptome sequencing data obtained from the cells with a knockdown of mRNA processing-related hits was compared with those of control siRNA-transfected cells, and the resulting heatmap showed a decrease of the expression levels of primary cilia disassembly-related genes including AURKA, CCNB1, NEK2, and PLK1 in the knockdown cells. D. The reduced RNA expression levels of the cilia disassembly regulators that were putatively targeted by the mRNA processing hits were confirmed by qRT-PCR. E. A proposed model for the essential roles of mRNA processing regulators in coupling of ciliary resorption with cell cycle re-entry. One-way ANOVA: ***P b 0.001 (A, B). The data are presented as the mean ± SEM (n = 3, [A]). Insets: representative images of each knockdown at higher magnification. Scale bars, 10 μM(A). activity of cilia disassembly regulators such as AURKA and PLK1 than may be required for the function of UPS to break down cilia assembly their expression is required to link ciliary resorption to the G1 → Stran- regulators in the G1 → S transition of cell cycle re-entry, thereby im- sition. Alternatively, we hypothesized that cilia disassembly regula- plying a possible role of cilia disassembly regulators in suppressing tors, activated by the mRNA processing mechanism, might be the activity of cilia assembly regulators (Fig. 6C). required to promote UPS-dependent proteolysis of cilia assembly regulators. To address this, we examined the expression levels of 3. Discussion cilia assembly proteins such as CPAP, KATANIN, and IFT88 in the mRNA processing hit-depleted SEMG cells, and found that the pro- The ciliary dynamics undergoing assembly and disassembly during tein levels (especially IFT88) in the knockdown cells were higher cell cycle progression is the subject of a long-standing debate regarding than those of controls 18 h after serum re-addition (Fig. 6A, B). In whether the primary cilium functions as a cell cycle checkpoint. The conclusion, these data suggest that the mRNA processing mechanism mechanism governing the link between ciliogenesis and cell cycle 1314 J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318 A C *** Serum - Serum + (18hr) 50 mCherry-Geminin Ctrl Ctrl PSMB3 PSMD1 PSMD8 PSMD11 MG132 40 siRNAs Smo-EGFP KDa 30 CPAP 140 20 Cells (%) KATANIN 10 50 100 0 IFT88 Ctrl PSMB3 PSMD1 PSMD8 PSMD11 MG132 siRNAs Actin Ctrl siRNA (+) MG132 (+) D 1.8 CPAP 1.6 IFT88 1.4 KATANIN 1.2 1 0.8 0.6 PSMB3 siRNA (+) PSMD1 siRNA (+) 0.4 0.2 Relative protein expression Relative protein change) (fold 0 Ctrl Ctrl PSMB3 PSMD1 PSMD8 PSMD11 MG132 Serum - Serum + E Serum - Serum + (2hr) PSMD8 siRNA (+) PSMD11 siRNA (+) Ctrl Ctrl PSMB3 PSMD1 PSMD8 PSMD11 MG132 siRNAs KDa HEF1 100

100 MPS1

Actin

B *** G0/G1 100 F S 3.5 HEF1 95 G2/M 90 3 MPS1 85 2.5 80 2 75 70 1.5 Cells (%) 65 1 60 0.5 20 Relative protein expression Relative protein change) (fold 0 0 Ctrl Ctrl PSMB3 PSMD1 PSMD8 PSMD11 MG132 (siRNAs) Serum - Serum + Ctrl PSMB3 PSMD1 PSMD8 MG132 PSMD11

Fig. 5. Proteasome-dependent decay of cilia assembly regulators is necessary for cilia disassembly and cell cycle re-entry. A. The SEMG cells were silenced with siRNAs targeting proteasome subunits or treated with MG132 and were analyzed by fluorescent imaging analysis 18 h after cessation of serum starvation. The cells showed inhibition of cilia disassembly and cell cycle re-entry. B. The cell cycle state of the knockdown or MG132-treated cells was analyzed by FACS and compared to that of controls 18 h after addition of serum. The quantified data were presented in the graph. C. The protein levels of cilia assembly regulators, including CPAP, KATANIN, and IFT88, in the proteasome subunit-depleted or MG132-treated cells were compared to those of control cells by Western blot assay. The protein levels of control cells were decreased, while those of knockdown and MG132-treated cells were high 18 h after cessation of serum starvation. D. The fold change of protein expression levels of cilia assembly regulators were quantified. E. The expression levels of cilia disassembly-related proteins including HEF1 and MPS1 in the proteasome subunit-depleted or MG132-treated cells were compared to those of control cells by western blot assay. The protein levels of control cells were slightly increased 2 h after serum re-addition, while those of knockdown and MG132-treated cells were much higher than controls. F. The fold change in protein expression levels of cilia disassembly regulators was quantified. The data are presented as the mean ± SEM (n = 4, [A, B]). One-way ANOVA: ***P b 0.001 (A, B). Insets: representative images of each knockdown at higher magnification. Scale bars, 10 μM(A). progression has been poorly understood. In the present study, we used a UPS-dependent proteolysis of cilia assembly regulators are essential for whole-genome RNAi screening to uncover the underlying mechanism a link between ciliary biogenesis and cell cycle control. that connects ciliogenesis to cell cycle progression. The machineries asso- It has recently been suggested that UPS is one of the core mecha- ciated with mRNA processing and the UPS were identified as the screen nisms in regulating ciliogenesis as well as cell cycle progression. For ex- hits, and follow-up functional assays suggested that the screen hits are ample, cilia-related roles of Anaphase-promoting Complex (APC), required for ciliary resorption and the G1 → S transition in cell cycle which is a well-known ubiquitin E3 ligase in cell cycle control, have re-entry. In particular, we demonstrated that the mRNA processing- been suggested [40]. To induce cilia disassembly, APC/CCDC20 is activated mediated expression/activity of cilia disassembly regulators and the when cells exit from the quiescent state [41]. In contrast, APC/CCDH1 J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318 1315

A B

Serum + (18hr) Ctrl CRNKL1 EIF3E PRPF6 PRPF8 2.5 CPAP siRNAs KATANIN KDa 2 IFT88 CPAP 140 1.5

KATANIN 1 50 100 IFT88 0.5 Relative protein expression Relative protein change) (fold 0 Actin 40 Ctrl CRNKL1 EIF3E PRPF6 PRPF8

C Cilia disassembly in G1 S

Pre-mRNA AAAA... mRNA processing regulators mRNA AAAA... UPS

Cilia disassembly regulators

Cilia assembly regulators

G0/G1 S

Fig. 6. A proposed model for the role of screen hits in a link between ciliogenesis and cell cycle progression. A. The protein levels of cilia assembly regulators including CPAP, KATANIN, and IFT88 in the mRNA processing-deficient cells were compared to those of control cells by western blot assay. The protein levels of knockdown cells were higher than those of control cells 18 h after cessation of serum starvation. B. The fold change of protein expression levels of cilia assembly regulators were quantified. C. The schematic diagram shows possible roles of screen hits including mRNA processing and UPS regulators in cilia disassembly and G1–S transition.

is necessary for cilia assembly by ensuring the optimal level of ciliary proteolysis of cilia assembly regulators is a stimulatory mechanism of proteins, such as Dishevelled, are present when cells enter the quiescent ciliary resorption in the G1 phase. state [42]. Along with APCs, Cul3-RING ubiquitin ligases are involved in Because mechanisms to regulate ciliogenesis and cell cycle have governing ciliogenesis by targeting ciliary proteins, such as Trichoplein been associated with several diseases, including , our results [13]. Although these data have indicated the importance of UPS in the have prompted us to explore an involvement of the screen hits in dynamic profile of cilia, the role of the proteasome complex in cancer. We therefore analyzed whole-exome sequencing data in- ciliogenesis has been poorly studied. Nevertheless, MG132-treated cluding public databases and our own samples from several types SEMG cells showed both defects in cilia assembly and disassembly, of and found considerable mutation frequencies in various implying a definite function of the proteasome in ciliary dynamics cancers for most of the hit genes (Supplementary Fig. S9). In conclu- (Supplementary Fig. S8). Furthermore, it has been recently reported sion, our findings suggest that a better understanding of mRNA that several subunits of the proteasome are localized at the base of processing- and UPS-dependent mechanisms to connect ciliogenesis cilia, and their activity is essential for cilia-mediate signal transduc- to the cell cycle progression may help to identify new therapeutic tion [43].Therefore,ourfindings not only support the previous re- targets in cancers. sults showing that UPS is necessary for cilia biogenesis but also contribute to a comprehensive understanding of the role of the pro- 4. Methods teasome in governing a connection between ciliogenesis and cell cycle progression. 4.1. Whole-genome siRNA library screening Although it has been widely accepted that the cilium is disassembled in two waves, in the G1 and S phases, in RPE1 cells, the regulatory mech- 4.1.1. The primary screening anism has been unknown [22,35]. In our study, abnormally increased An arrayed library containing pooled siRNAs targeting 18,055 levels of HEF1, MPS1, and cilia assembly regulators, such as CPAP, human genes (GE Healthcare Dharmacon Inc.) was screened in KATATIN, and IFT88 in the G1 phase inhibited cilia disassembly and duplicate. Assay plates (384-well plates with an optical bottoms; cell cycle re-entry in the UPS deficient cells (Fig. 5). Therefore, these re- Greiner) were spotted with 1 μlof0.5μM siRNA using the Velocity sults implied that cilia assembly regulators might suppress HEF1 and 11-Bravo Pipettor with a 384 ST head. Reverse transfection was per- MPS1; thus, the failed decay of cilia assembly proteins might interfere formed using Lipofectamine RNAiMAX with a final siRNA concentra- with the role of HEF1 and MPS1 that control cilia disassembly and cell tion of 10 nM. SEMG cells were resuspended in DMEM/F12 cycle re-entry. Therefore, our findings suggest that the UPS-mediated supplemented with 10% FBS and seeded in assay plates using the 1316 J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318

Matrix-well Mate (2000 cells in 40 μlmediumineachwell).Thecul- ciliogenesis-coupled cell cycle. For functional studies, individual oligos ture medium was replaced with DMEM 24 h after the transfection of Dharmacon ON-TARGET-PLUS siRNAs were used at a final concentra- using the TiterTek-MAP-C, and the cells were incubated for another tion of 20 nM. 48 h before fixation. 4.4. siRNA transfections 4.1.2. Imaging and image analysis Imaging related to the siRNA screening was performed on the Opera Reverse transfection procedures were set up using Lipofectamine® QEHS system (Perkin Elmer). Six fields were photographed in each well RNAiMAX (Invitrogen). The final siRNA concentration was 20 nM, and at wavelengths of three dyes: DAPI, Cy3, and Cy5. Acapella 2.0 software SEMG cells were resuspended in DMEM/F12 supplemented with 10% (Perkin Elmer) was used to perform image segmentation and cytometry FBS and then seeded on Lab-Tek™ 8-well chamber slides (Nunc). The with algorithms similar to those previously described by Kim et al. [44]. siRNAs were diluted in Transfectagro™ (Corning) and mixed with Plate-to-plate variability was normalized using a control-based meth- Lipofectamine® RNAiMAX in Transfectagro™ at the 1:1 ratio by volume. od; the associated control samples were aggregated, and the mean The medium was refreshed for the transfected cells after 6 h of incuba- and variance across the wells were determined. The Z-score for all tion, and the cells were assayed after 48–72 h, according to the experi- wells with a siRNA knockdown was calculated using the mean and var- mental conditions. iance from the control data, and the final results were obtained using a threshold of more than two standard deviations (N2 SD). Additional in- 4.5. Spliceostatin A (SSA) treatment valid data that were ascribed to cell toxicity or out-of-focus images were excluded through manual inspection using the CyteSeer 2.7 software. SSA (AdooQ Bioscience, cat. # A12700), which as dissolved in 0.1% dimethyl sulfoxide (DMSO) was diluted in a cell culture media 4.1.3. Confirmation screening (DMEM/F12 supplemented with 10% FBS, 1% penicillin streptomycin). During this procedure, re-arrayed siRNAs targeting a total of 1481 The cells, plated on Lab-Tek™ 8-well chamber slides (Nunc) were treat- genes (Group A hits from the primary screening) were tested in tripli- ed with 50 ng/ml of SSA for 24 h. cate. SEMG cells (1300 cells for the serum-containing medium and 1850 cells for the serum-free medium in 40 μl of the medium for each 4.6. Image analysis well) were transfected and incubated with or without serum for 24 h before fixation. The confirmation screening was based on measurement After the siRNA transfection, the SEMG cells at 90–100% confluence of the percentage of ciliated cycling cells under serum-free conditions. were fixed with 4% paraformaldehyde (PFA) and stained with DAPI. For plate normalization, the quantification data were converted to a To monitor the primary cilia and cell cycle state, we used an LSM700 Z-score using unique non-targeting siRNAs included in each plate as ref- confocal microscope (Carl Zeiss). The total numbers of ciliated cells erence data points. Genes were defined as confirmed screen hits if the (green) and cycling cells expressing Geminin (red) were counted genes selected with a cut-off of N2 SD were subsequently validated by using the Image J software (NIH) and were analyzed using one-way manual image analysis in the CyteSeer 2.7 software. analysis of variance (ANOVA) followed by independent Tukey's post hoc test using SigmaPlot 12 for at least three independent experiments. 4.1.4. Hit classification To classify the primary screen hits, the apparent knockdown pheno- 4.7. Cell cycle analysis types were compared to those of control cells with cilia by maintaining the quiescent state via serum starvation. Three groups were distin- siRNA transfection was performed on SEMG cells seeded in 100 mm guished according to changed ciliogenesis and/or cell cycle features on dishes and grown to approximately 70% confluence. The cells were the basis of measurement of the percentage of ciliated or cycling cells: trypsinized and fixed in 70% ethanol, followed by incubation with Group A, formation of cilia in cycling cells; Group B, reduction of cilia 100 g/ml RNase A (Mentos) for 15 min at room temperature. To deter- in quiescent cells; and Group C, decreased number of ciliated cells and mine cell cycle state of each cell, the cells were incubated with 50 g/ml increased number of proliferating cells in the condition of quiescence. propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature, and the DNA contents were analyzed by FACS anal- 4.2. Cell culture ysis using a BD FACSVerse™ flow cytometer and BD FACSuite software (Becton Dickinson). The statistical analysis involved one-way ANOVA hTERT-RPE1 cells were cultured in the DMEM/F12 medium supple- followed by an independent Tukey's post hoc test using SigmaPlot 12 mented with 10% FBS under standard conditions (37 °C, 5% CO2). Plas- for at least three independent experiments. mid DNAs carrying mouse Smo-EGFP and mCherry-Geminin (1–110) fusion genes were transfected into hTERT-RPE1 cells, and the stable 4.8. Whole-transcriptome sequencing cell line Smo-EGFP-mCherry-Geminin/hTERT-RPE1 (SEMG), was established via G418 selection. To induce ciliogenesis, the cells were SEMG cells were cultured in 6-well plates (Corning) in a serum- serum-starved in the serum-free DMEM/F12 medium for 24–48 h containing (FBS 10%) or serum-free (FBS 0%) medium after siRNA trans- before fixation. fection and were harvested 72 h later. The cells were washed with PBS, and total RNA was extracted using the RNA extraction kit (Qiagen). RNA 4.3. siRNA reagents was tested for quality and yield using a NanoDrop 1000 spectrophotom- eter and an Agilent 2100 Bioanalyzer. cDNA was synthesized from the A Dharmacon human ON-TARGET-PLUS pooled siRNA library (GE total RNA according to the standard protocol of Illumina Inc. for high- Healthcare Dharmacon Inc.) was used, in a standard 384-well assay throughput sequencing, and the transcriptome was sequenced using plate with an optical bottom (Greiner) using the Velocity 11-Bravo Illumina Hiseq2000. The reads from the FASTQ files were mapped Pipettor with a 384 ST head. Each gene was targeted using a pool of against the hg19 human reference genome using TopHat version 2.0.6 four duplexes per well, and 18,055 genes of the entire library targeted (http://tophat.cbcb.umd/edu/). The output files in BAM format were an- a total of 18,301 human genes in the initial screening procedure. The alyzed in Cufflinks version 2.1.1 (http://cufflinks.cbcb.umd.edu/) to esti- pooled siRNAs targeting KIF3A were used as a positive control for the mate the transcript abundance and assemble transcripts. The transcript transfection, and AURKA, NEK1, BUB1B,andOFD1 siRNAs were included assembly was compared with reference annotation Ensemble GTF in the in the confirmation screening plates as positive controls for effects on Cuffcompare software, and the differentially expressed genes were J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318 1317 extracted using Cuffdiff with the following criteria: q-value b0.05 and a E3 medium containing 0.2 mM N-phenylthiourea (PTU; Sigma-Aldrich 1.5-fold change. The heatmap of genes with expression levels signifi- Chemistry, cat. # P7629). cantly different between control and siRNA knockdown groups was generated using the heatmap.2 feature of the gplots R package (http:// 4.11.2. Microinjection with morpholino oligos (MOs) CRAN.R-project.org/package=gplots), with the mRNA abundance Translation-blocking MOs were designed and synthesized by Gene expressed in FPKM (fragments per kilobase of exon per million reads Tools (Philomath, OR 97370, USA). Each MO was diluted in distilled mapped). water at a concentration of 4–5 μg/μl and then injected into the yolk of zebrafish embryos at one to four cell stages using a gas-used microinjec- 4.9. Quantitative real-time PCR tion system (PV83 Pneumatic PicoPump, SYS-PV830, World Precision Instruments, USA). The following MO sequences were used for the

The reaction of reverse transcription (RT) with oligo[dT]20 microinjection: primers (Invitrogen) and 1 μgoftotalRNAasatemplatewasper- formed using SuperScript III reverse transcriptase (Invitrogen). prpf8 ATG-MO, 5′-AAAGCAGTTTCTCGCCAGTGACTGT-3′. The RT-generated cDNAs were used as the templates, and the psmd8 ATG-MO, 5′-CAGTCTCTTTCAACACAGACGCCAT-3′. Power SYBR Green PCR Master Mix (Applied Biosystems) and control MO, 5′-CCTCTTACCTCAGTTACAATTTATA-3′. primers designed for each gene (listed below) were added to each reaction mixture for qRT-PCR. To prevent formation of primer 4.11.3. Microinjection with mRNA dimers, PCR conditions were optimized, and the results were sub- The full-length zebrafish psmd8 was amplified by RT-PCR using sequently confirmed by analyzing amplimer dissociation curves zebrafish cDNA, which was synthesized with AccuPower® Rocket Script after quantitative PCR. The qRT-PCR reactions were conducted RT Premix (K-2101, Bioneer, Korea) from total RNA, extracted from using a 7300 Real-time PCR System (Applied Biosystems); a consti- zebrafish embryos at 24 hpf using the Accuzol™ Total RNA Extraction tutively expressed , GAPDH,servedasthebio- Reagent (K-3090, Bioneer, Korea). The zebrafish psmd8 cDNA was massreference.ThefollowingprimerswereusedfortheqRT-PCR: subcloned into pCS2+ (Clontech) and the Capped mRNA was synthe- sized using the mMESSAGE mMACHINE® SP6 Kit (AM1340, Thermo CCNB1_F,5′- ACATGGTGCACTTTCCTCCT- 3′. Fisher Scientific). The zebrafish psmd8 mRNA (200 pg/nl) was injected CCNB1_R,5′- AGGTAATGTTGTAGAGTTGGTGTCC- 3′. into the psmd8 MO-injected embryos. The following primer sequences NEK2_F,5′- CATTGGCACAGGCTCCTAC- 3′. were used for the RT-PCR: NEK2_R,5′- TGGAGCCATAGTCAAGTTCTTTC- 3′. ′ ′ AURKA_F,5′- GCAGATTTTGGGTGGTCAGT- 3′. Forward primer, 5 -CCGGAATTCATGGGCGTCTTGTTGAAAGAG-3 , AURKA_R,5′- TAGTCCAGGGTGCCACAGA- 3′. Reverse primer, 5′-GCGCGTCTAGACTACACAATCATTTCCAGCTGTCTT PLK1_F,5′- CACAGTGTCAATGCCTCCA- 3′. GC-3′. PLK1_R,5′- TTGCTGACCCAGAAGATGG- 3′. 4.11.4. Whole-mount immunostaining of zebrafish larvae 4.10. Western blotting To induce ciliary defects, we treated zebrafish embryos with either 40 μM SSA (AdooQ Bioscience, cat. # A12700) or 50 μM MG132 The proteins of SEMG cells were separated on 10% SDS-PAGE gels (Calbiochem, cat. # 474,790) at 24 hpf (Supplementary Fig. S3B), and (we loaded 30 μg of protein per lane), followed by transfer onto nitro- the control embryos were treated with 1% DMSO. The drug-treated em- cellulose membranes (GE Healthcare). After incubation in blocking bryos were raised at 28.5 °C until 3 days post-fertilization (dpf) and buffer (5% skim milk in TBST [Tris-buffered saline pH 7.5 with 0.5% fixed in Dent's fixative (80% MeOH, 20% DMSO) at room temperature Tween-20]) for 1 h at room temperature, membranes were blotted for 3 h. The fixed zebrafish larvae were immunostained with an anti- with a mouse anti-Aurora A (cat. #12100, Cell Signaling), rabbit anti- GT335 antibody (AdipoGen, cat. # A20631002; 1:400 dilution) as a pri- CPAP (cat. #11517-I-AP, Proteintech), rabbit anti-IFT88 (cat. #13967- mary antibody at 4 °C overnight and with a goat anti-mouse Alexa I-AP, Proteintech), mouse anti-MPS1 (cat. #ab11108, Abcam), mouse Fluor® 488-conjugated antibody (Invitrogen, cat. # A11001, 1:1000) anti-PLK1 (cat. #ab17057, Abcam), mouse anti-HEF1 (cat. #ab18056, as a secondary antibody at room temperature for 2 h in a half-diluted Abcam), mouse anti-beta-Actin (C4) (cat. #sc-47778, Santa Cruz Bio- blocking solution (10% normal goat serum, 0.5% Tween20 in PBS) and technology), and mouse anti-Katanin p60 (cat. #sc-373814, Santa 12 μM DAPI (Molecular probes, cat. #D3571) at room temperature for Cruz Biogechnology) antibodies overnight at 4 °C. The membranes 30 min in a PBST (0.5% Tween 20 in PBS). The fluorescent images were then washed two times with TBST and incubated with secondary were obtained using a LSM700 confocal microscope (Carl Zeiss). antibodies: a goat anti-mouse IgG horseradish peroxidase (HRP)-conju- gated antibody (cat. #sc-2031, Santa Cruz Biotechnology) and a goat Author contributions anti-rabbit IgG HRP-conjugated antibody (cat. #sc-2030, Santa Cruz) for 1 h at room temperature. After washing the membranes two times J.H.K. and S.M.K. performed molecular biological and cell experi- with TBST, we visualized the bands using the ECL detection system ments and the in vivo experiments on zebrafish. J.K. designed and per- (Bio-Rad). formed the primary siRNA screening, with assistance from P.A.-B. on screening and from S. H.-G. on scoring. E.S. performed statistical analysis 4.11. In vivo experiments of the screening results. J.E.L. and J.G.G. wrote the manuscript and de- signed the experiments with substantial contributions from J.-G.J. and 4.11.1. Zebrafish housing and manipulations C.H.K. Adult zebrafish were maintained at 28.5 °C and pH 7.0–7.9 with a Supplementary data to this article can be found online at http://dx. cycle of light (13 h) and dark (11 h) per day in the automatic system doi.org/10.1016/j.bbamcr.2016.03.021. (Constructed by Genomic-Design, Korea). The zebrafish embryos were collected by natural breeding and incubated in the E3 medium Transparency document

(297.7 mM NaCl, 10.7 mM KCl, 26.1 mM CaCl2, and 24.1 mM MgCl2) containing 1% methylene blue (Sigma-Aldrich) at 28.5 °C. To inhibit for- The Transparency document associated with this article can be mation of melanin, we raised the zebrafish larvae (after 24 hpf) in the found in the online version. 1318 J.H. Kim et al. / Biochimica et Biophysica Acta 1863 (2016) 1307–1318

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