The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells

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Michael Ignarski,1 Constantin Rill,1 Rainer W.J. Kaiser,1 Madlen Kaldirim,1 René Neuhaus,1 Reza Esmaillie,1 Xinping Li,2 Corinna Klein,3 Katrin Bohl,1 Maike Petersen,1 Christian K. Frese,3 Martin Höhne ,1 Ilian Atanassov,2 Markus M. Rinschen,1 Katja Höpker,1 Bernhard Schermer,1,4,5 Thomas Benzing,1,4,5 Christoph Dieterich,6,7 Francesca Fabretti,1 and Roman-Ulrich Müller 1,4,5

1Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany; 2Proteomics Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany; 3Proteomics Facility, Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases, 4Nephrolab, Cologne Excellence Cluster on Cellular Stress Responses in Aging- associated Diseases, Faculty of Medicine and University Hospital Cologne, and 5Systems Biology of Ageing Cologne, University of Cologne, Cologne, Germany; 6Department of Internal Medicine III, Klaus Tschira Institute for Integrative Computational Cardiology, University Hospital Heidelberg, Heidelberg, Germany; and 7German Center for Cardiovascular Research (DZHK)–Partner site, Heidelberg/Mannheim, Germany

ABSTRACT Background RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically. Methods We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA‐binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome. Results We identified .1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia. Conclusions These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia.

J Am Soc Nephrol 30: 564–576, 2019. doi: https://doi.org/10.1681/ASN.2018090914

Received September 9, 2018. Accepted January 20, 2019. Internal Medicine, and Center for Molecular Medicine Cologne, University of Cologne, Kerpener Str. 62, 50937 Köln, Germany. M.I., C.R., F.F., and R.-U.M. contributed equally to this work. Email: [email protected]

Correspondence: Dr. Roman-Ulrich Müller, Department II of

564 ISSN : 1046-6673/3004-564 J Am Soc Nephrol 30: 564–576, 2019 www.jasn.org BASIC RESEARCH

RNA metabolism is closely regulated by a group of special- Significance Statement ized proteins that are able to directly interact with RNA. RNA-binding proteins (RBPs) influence bound transcripts RNA-binding proteins (RBPs) are crucial regulators of cellular bi- starting with their biosynthesis and have a significant effect ology, and recent evidence suggests that regulation of RBPs that on RNA stability, translation rate, and the velocity of deg- modulate both RNA stability and translation may have a profound effect on the proteome. However, little is known about regulation of radation. This effect on the transcriptome consecutively RBPs upon clinically relevant changes of the cellular microenviron- affects the proteome and thereby puts RBPs at the center ment. The authors used high-throughput approaches to study the of regulation for various signaling pathways, metabolism, cellular RNA‐binding proteome in differentiated tubular epithelial and cell fate.1 Whereas in the past RBPs were assumed to cells exposed to hypoxia. They identified a number of novel RBPs fi primarily contain classic RNA-binding domains (RBDs), (suggesting that these proteins may be speci c RBPs in differentiated tubular epithelial cells), and found quantitative differences in 1 recent work has challenged this view. As an example, RBP-binding to mRNA associated with hypoxia versus normoxia. RNA interactome capture (RIC) approaches in both hepatic These findings demonstrate the regulation of RBPs through envi- and cardiac cells have shown the unexpected potential of cel- ronmental stimuli and provide insight into the biology of hypoxia- lular metabolic enzymes to have RNA-binding capacity with- response signaling in the kidney. out disposing of classic RBDs.2,3 Furthermore, a subset of — RBPs is capable of binding both RNA and DNA thereby 0.05% Trypsin. Cells were tested for mycoplasma contamina- fl in uencing replication, transcription rate, and the response tion using a PCR Mycoplasma Test Kit I/C (Venor GeM; 4 to DNA damage. This increasing knowledge of protein-RNA Sigma). Transfections were carried out in 60%–80% confluent interactions has been greatly facilitated by the development cells using calcium phosphate13 or lipofection (Lipofectamine — fi of a number of novel techniques allowing for the identi - 2000; Thermo Scientific) according to the manufacturers’ in- — cation of RBPs and their target transcripts that are structions. mIMCD-3 cells were differentiated using serum mostly based on of UV-induced crosslinking and RNA- starvation as described previously.14,15 protein precipitation coupled with mass spectrometry and RNA sequencing.5 In recent years, these techniques have Antibodies been employed in cells from a number of different tissues1; Epitope/Name Manufacturer ID however, little is known about the global RNA-binding land- FLAG (M2) Sigma-Aldrich F1804–1MG scape in kidney cells. Nonetheless, several targeted studies have GFP (B2) SantaCruz BioTech sc-9996 shown the general potential of RBPs to affect the cellular Hif1a Cayman Chemical 10006421 biology of kidney cells. One of the most extensively studied Pericentrin Abcam AB4448 RBPs, HuR, has been implicated as a key player in the in- b-tubulin (E7) Developmental Studies E7 duction of renal fibrosis and inflammation both in vitro and Hybridoma Bank in vivo,6 and could be shown to bind Hif1a mRNA thereby Acetylated tubulin Sigma-Aldrich T6793 increasing both its stability and translation rate.7 This find- MPDZ SantaCruz BioTech sc-136293 ing is of special interest taking into account the low oxygen KIF13B Developmental Studies AFFN-KIF13B-7H5 tensionintherenalmedullaandtheroleofhypoxiasignal- Hybridoma Bank ing in both AKI and CKD.8,9 Seeing that hypoxia leads to a ARL13B Proteintech 17711–1-AP general stop in protein production and a shift toward pref- For a list of secondary antibodies see the Supplemental Meth- erential translation of proteins required for the cellular re- ods section. sponse to hypoxia, it is likely that RBPs play a major role in the regulation of the hypoxia-associated transcriptome and Plasmids and Cloning proteome.10,11 In order to extend the knowledge toward a All plasmids used in this study were generated using restriction more global understanding of the RNA-protein interactome enzyme cloning, modified using QuikChange Mutagenesis in the kidney, we employed the RIC approach12—for the (Stratagene), or were purchased from Addgene. For a detailed first time—in ciliated cells derived from the inner medul- list of plasmids and primers used in this study see the Supple- lary collecting duct (mIMCD-3) and characterize the mod- mental Methods section. ulation of the RBPome by hypoxia. TALEN-Based Transgenesis Stably integrated transgenic cell lines were generated using METHODS TALEN technology by cotransfecting TALEN-encoding plasmids specific for the human AAVS1 locus, as previously de- Cell Culture and Transfection scribed.16,17 Starting 24 hours after transfection, cell lines were Human HEK 293Tand murine mIMCD-3 cells were obtained steadily selected with 2 mM Puromycin. All generated trans- from ATCC and grown in standard media (+10% FBS; DMEM genic cell lines were genotyped by integration PCR (for details for HEK293T, DMEM-F12 containing 2 mM Glutamax for see the Supplemental Methods section) and characterized by mIMCD-3) at 37°C, 5% CO2, and routinely passaged using western blot and fluorescence microscopy.

J Am Soc Nephrol 30: 564–576, 2019 Kidney Epithelial RNA-Binding Proteome 565 BASIC RESEARCH www.jasn.org mRNA Interactome Capture of Ciliated mIMCD-3 Cells Immunofluorescence Microscopy Oligo (dT) capture and isolation of protein-RNA complexes Cells grown on cover slips were washed once with PBS sup- were performed as previously described.18 Briefly, for each plemented with Ca2+/Mg2+ (PBS+), fixed with 4% PFA solu- condition, ten cell culture dishes with 15 cm diameter were tion at RT for 10 minutes, and rinsed twice with PBS+. Cells used. After reaching a confluency of about 70%, cells were were then blocked in 5% donkey serum and 0.1% Triton serum starved to induce differentiation and consequently cil- X-100 in PBS at RT and incubated in primary antibody at iation for a total duration of 30 hours. During starvation, and RT for 1 hour. After rinsing the cells three times, secondary before hypoxia treatment, the cells were incubated with antibody was added for 1 hour at RT, after which the stained 200 mM 4-thiouridine (4-SU) for a total of 18 hours to met- cells were mounted on glass slides with 10 ml ProLong Gold abolically label RNA. Six hours before harvesting, 50% of the mounting medium with DAPI to visualize nuclei. Images were dishes were transferred to a hypoxia cell culture incubator acquired using an Axiovert 200M fluorescence microscope (x63/ (1% O2). After crosslinking with UVA (365 nm), cells were 1.2W) equipped with the Axiocam MRm, supporting the Apo- harvested and lysed followed by an oligo (dT) pulldown. RNP Tome system and Axiovision 4.8 software or ZEN software complexes were treated with RNase/benzonase and submitted (Zeiss). Unless indicated otherwise, all immunofluorescence to mass spectrometry after a trypsin digest. The details of these microscopy was performed in cells kept in normoxia. procedures are provided in the Supplemental Methods section. Polynucleotide Kinase Assay HEK293T cells transfected with pcDNA6 expressing triple Generation of Cell Lysates for Proteome Analysis FLAG-tagged genesof interestwereUVC(254nm)crosslinked, The whole proteome of mIMCD-3 grown under normoxic resuspended in lysis buffer (100 mM KCL, 5 mM MgCl2, or hypoxic conditions was measured in three biologic repli- 10 mM Tris pH 7.5%, 0.5% NP40, 1 mM DTT, protease in- cates. Approximately 106 ciliated mIMCD-3 cells were washed hibitors), and homogenized through a 23G needle on ice. For with PBS, centrifuged (500 3 g, 5 minutes, 4°C), snap-frozen, nuclear proteins, samples were additionally sonicated (Bio- and resuspended in 300 ml 8 M urea. Lysates were sonicated rupter Pico; ten intervals: 30 seconds on/off). Lysates were (Ultrasonics Sonifier) and protein concentration was mea- cleared by centrifugation (20,000 3 g) and treated with sured using the Pierce BCA Protein Assay Kit. Equal amounts 2 U/ml Turbo DNase (Invitrogen) and 40 U/ml RNase I (Am- of total protein were reduced with 50 mM DTT and alkylated bion) for 15 minutes at 37°C. Immunoprecipitation was per- in the dark using 1:5 total volume of IAA buffer for 1 hour at formed with anti-FLAG M2 (Sigma) coupled to Protein G room temperature. In order to lower the urea concentration Dynabeads (ThermoFisher) for 2 hours at 4°C. Beads were to ,2 M, each sample was diluted with 50 mM ammo- washed five times with washing buffer (500 mM NaCl, nium bicarbonate. Finally, samples were digested at RT over- 20 mM Tris pH 7.5, 1 mM MgCl2, 0.05% NP40) and twice night with trypsin, stage-tipped, and submitted to mass with polynucleotide kinase (PNK) buffer (50 mM Tris spectrometry. pH 7.5, 50 mM NaCl, 10 mM MgCl2,0.5%NP40).Beads were resuspended in PNK buffer containing 5 mM DTT, Mass Spectrometry 0.2 mCi/ml(g32P)ATP (Hartmann-Analytic), and 1 U/mlT4 Peptides were separated on a 25-cm long, 75-mminternal PNK (ThermoFisher). The labeling was carried out for diameter PicoFrit analytic column (New Objective) packed 20 minutes at 37°C. Beads were washed five times with PNK with a 1.9 mm ReproSil-Pur 120 C18-AQ media (Dr. Maisch), buffer and boiled with laemmli buffer. The samples were re- using EASY-nLC 1000 (ThermoFisher Scientific). The col- solved on 4%–12%bis-trisgels(ThermoFisher)andtrans- umn was maintained at 50°C. Buffers A and B were 0.1% ferred onto Protran Nitrocellulose membrane (Schleicher formic acid in water and 0.1% formic acid in acetonitrile, and Schuell). The blot was exposed to a storage phosphor respectively. Peptides were separated on a segmented gradi- screen (Amersham) and the signal was detected with a Ty- ent from 2% to 5% buffer B for 10 minutes, and from 5% to phoon scanner (GE Healthcare). IP efficiency was controlled 20% buffer B for 100 minutes at 200 nl/min. Eluting peptides by western blotting with anti-FLAG M2 (Sigma). All PNK were analyzed on a QExactive Plus mass spectrometer (Ther- assays were performed using cells kept under normoxic moFisher Scientific). Peptide precursor mass-to-charge ratio conditions. (m/z) measurements (MS1) were carried out at 70,000 resolution in the 300–1800 m/z range. The top ten most intense Data Analysis, Statistics, and Data Sharing precursors with charge states from 2 to 7 only were selected Statistical analysis of both the whole proteome and the RNA for HCD fragmentation using 25% normalized collision en- interactome raw data were performed with Perseus software,19 ergy. The m/z values of the peptide fragments were measured version 1.6.0.7. Default parameters (only identified by site, re- at 17,500 resolution using an AGC target of 2e5 and 80-ms verse, and contaminants) were used for filtering the dataset. Raw maximum injection time and 4.0% underfill ratio. Upon frag- data and MaxQuant20 (version 1.5.3.8) output were uploaded to mentation, precursors were put on a dynamic exclusion list the PRIDE repository (http://www.ebi.ac.uk/pride; project ac- for 45 seconds. cession: PXD010530). More details regarding the proteomics

566 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 564–576, 2019 www.jasn.org BASIC RESEARCH

A Crosslinked RBP x x x UVA x 30 hours starvation x Nucleus x x + Poly-A RNA 18 hours labeling AAAAAA 6 hours - Oligo-dT capture mIMCD-3 cells 4-SU 1% O2 x x 12 hours 12 hourss x x x 6 hours x 70% confluency ciliated cells + x 21% O2 AAAAAA oligo-dT beads TTTTTT - RNase treatment x x x x x x x

Mass spectrometry Data analysis

B C D

7 known RBPs Hyp-CL Hyp+CL novel RBPs Norm-CL Norm+CL 6 others M -CL +CL NH NH 5 250 40 130 4 100 20 3 70 p value +CL vs -CL

0 10 2

55 -log -20 1 35 component 2(17.2 %) 0 25 -40 -20 0 20 40 60 -2 -1 0 1 2 3 4 5 6 7

component 1(45.3 %) log2 FC +CL vs -CL

Figure 1. RNA-interactome capture reveals hundreds of RNA-associated proteins in differentiated inner medullary collecting duct cells. (A) Culturing scheme depicting the treatment of mIMCD-3 cells and schematic representation of the RIC procedure. mIMCD-3 cells were grown to 70% confluency and starved by serum deprivation to induce differentiation. After 12 hours, the cell culture medium was supplemented with 4-SU. 4-SU labeling and starvation were continued for 12 hours before 20 of 40 dishes were transferred to hypoxic conditions (1% O2, blue) for 6 hours, whereas the other 20 dishes were kept under normoxic conditions (21% O2, pink). After this treatment ten dishes of each condition were UVA crosslinked (UVA+), whereas the other ten dishes re- mained noncrosslinked (UVA2). Aliquots of the collected cell lysates were used for proteome analysis and the rest were submitted to RIC. RIC: after crosslinking and cell lysis, polyadenylated transcripts were captured with oligo-dT beads. The captured RNA- protein complexes were RNase treated and putative RBPs were identified using quantitative label-free proteomics. (B) Analysis of protein enrichment after oligo (dT) capture and RNase treatment by silver staining. The enrichment of a complex protein pattern is only detectable in the lanes with UVA-irradiated samples (+CL) and is absent in the lanes with nonirradiated samples (2CL). Lanes with samples after hypoxic or normoxic treatment are indicated by N or H, respectively. M, molecular weight marker. (C) RNA-bound proteome (RBPome) data of mIMCD-3 cell samples. Principal component analysis of RIC (on the basis of iBAQ intensities) for crosslinked (+CL/red) and noncrosslinked (2CL/black) samples showing a clear separation of the two groups. Open circles represent samples with hypoxia treatment; filled circles represent samples kept under normoxic conditions. (D) Scatter plot of the t test comparison of protein abundance in the crosslinked and noncrosslinked samples. The x axisisthemeanlog2 differenceinabun- dance of crosslinked versus noncrosslinked samples; presented on the y axis are the corresponding 2log10 P values. The cutoff for significanceisanFDR,0.1. RBPs significantly enriched according to our cutoff are represented by large circles. RBPs not reaching significance are represented by small circles. Red, mouse RBPs and mouse orthologs of human RBPs previously described (summarized in Hentze et al.1 [2018]); black, novel RBPs; gray, others. FC, fold change; Hyp, hypoxia; Norm, normoxia; vs, versus.

J Am Soc Nephrol 30: 564–576, 2019 Kidney Epithelial RNA-Binding Proteome 567 BASIC RESEARCH www.jasn.org

A B 100% 46 94 mIMCDs mRNA interactome 90% (n=1058) 25 80%

175 69 70% 4338 789 60% 199 Homo sapiens Mus musculus other (n=1393) 50% (n=1914) mouse RBPs 614 464 964 336 40%

30%

20% 1695 10%

0% proteome class I class I and II (6033) (510) (1058)

Pfam C YT521-B-like domain (YTH) 100% D La domain (La) KH domain (KH_1) 90% RNA recognition motif (RRM_1) Helicase associated domain (HA2) 220 80% Oligonucleotide/oligosaccharide-binding (OB)-fold (OB_NTP_bind) DEAD/DEAH box helicase (DEAD) 70% SAP domain (SAP) Zinc finger C-x8-C-x5-C-x3-H type (zf-CCCH) 60% 521 Helicase conserved C-terminal domain (Helicase_C) 02468101214 50% enrichment factor

40% Smart 421 Domain in the RNA-binding Lupus La protein (LA) 30% Domain in DSRM or ZnF_C2H2 domain containing proteins (DZF) K homology RNA-binding domain (KH) 20% RNA recognition motif (RRM) Helicase associated domain (HA2) 151 10% Putative DNA-binding (bihelical) motif (SAP) zinc finger (ZnF_C3H1) 0% Helicase superfamily c-terminal domain (HELICc) RNA binding DNA binding DEAD-like helicases superfamily (DEXDc) (GO MF) (GO MF) 024681012 present just in proteome present in class I and II enrichment factor

E GO MF GO BP slim poly(A) RNA binding cytoplasmic translation poly-purine tract binding ribosomal small subunit biogenesis mRNA 5'-UTR binding rRNA processing poly(U) RNA binding ribosome biogenesis snoRNA binding ribosomal large subunit biogenesis poly-pyrimidine tract binding RNA splicing rRNA methyltransferase activity mRNA processing ATP-dependent RNA helicase activity RNA processing RNA helicase activity mRNA metabolic process RNA-dependent ATPase activity nucleobase-containing compound transport oxidoreductase activity ion transport protein serine/threonine kinase activity cellular lipid metabolic process substrate-specific transporter activity transmembrane transport protein complex binding secretion molecular transducer activity Golgi vesicle transport signal transducer activity endocytosis substrate-specific transmembrane transporter activity membrane invagination GTPase regulator activity alcohol metabolic process transmembrane transporter activity carbohydrate metabolic process nucleoside-triphosphatase regulator activity vesicle-mediated transport –5 –4 –3 –2 –1 0 1 2 3 4 –5 –4 –3 –2 –1 0 1 2 3 4 log enrichment factor log enrichment factor 2 2

Figure 2. RNA-interactome capture in mIMCD-3 cells clearly enriches for proteins with RNA-binding capacity. (A) Venn diagram depicting the number of RBPs identified in the mIMCD-3 RBPome (white) and the overlap with previously identified mouse (light gray) and human (dark gray) RBPs. Of 1058 RBPs identified in mIMCD-3 cells, 1033 were previously described in mouse (964) or human (858) samples (Hentze et al.1 [2018]). Twenty-five RBPs were not previously identified in mouse or human. (B) The fraction of known mouse RBPs1 contained in the mIMCD-3 proteome in comparison with the mIMCD-3 RBPome. Of 6033 proteins measured in the mIMCD-3 proteome, 1695 have been previously identified as mouse RBPs (28.1%). For the 510 class I RBPs, 464 have been previously observed (91%) and, among the combined class I and class II RBPs, 964 of 1058 (91.1%) have been previously identified. We calculate a 3.2-fold enrichment of class I and class I plus class II RBPs in the RBPome over the proteome. Gray, proteins previously identified as RBPs; white, not known to be RBPs (other). (C) Comparison of mIMCD-3 RBPome (gray) and mIMCD-3 proteome (white) for GO terms: “RNA- binding” and “DNA-binding.” (D) Pfam and Smart protein domains with significant enrichment in the mIMCD-3 RBPome. Statistical significance was determined with the Fisher exact test (Perseus software; FDR,0.05). (E) mIMCD-3 RBPome gene ontology enrichment

568 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 564–576, 2019 www.jasn.org BASIC RESEARCH analyses, the consecutive GO term/Pfam/Smart, and KEGG en- proteins that were not significantly enriched may still be richments as well as correlation analyses are provided in the RBPs. Consequently, a second class of RBPs—class II—was Supplemental Methods section. An interactive online database defined containing all proteins detected in our screen that had was created using the “shiny” package in “R” and is provided at previously been identified to show RNA-binding capacity http://shiny.cecad.uni-koeln.de:3838/mIMCD_RBPome. using a recently published compendium, which includes six datasets for Mus musculus and six for Homo sapiens.1 On the basis of these criteria a total of 1058 proteins fell into these two RESULTS classes of RBPs (510 class I, 548 class II; see Supplemental Table). These proteins are illustrated in the scatterplot in RIC to Identify the Global RBPome in Differentiated Figure 1D, depicting the enrichment in crosslinked samples Inner Medullary Collecting Duct Cells (FDR,0.1 for enrichment over noncrosslinked samples; Tostudy the RNA interactome in kidney epithelial cells we used s0=0.1) as well as the overlap with both the mouse RBPs and the photoactivatable nucleotide 4-SU for crosslinking of RNA- the murine orthologs of the human RBPs listed in the com- bound proteins.18,21 To this end, mIMCD-3 cells were grown pendium.1 This plot underlines the validity of the dataset be- to 70% confluency followed by starvation-induced differenti- cause the vast majority of proteins captured had been ation for 12 hours and subsequent 4-SU labeling (Figure 1A). described to bind to RNA previously (Figure 1D). In order Six of 12 samples (three crosslinked and noncrosslinked, re- to provide a user-friendly platform to query our data, an on- spectively) were exposed to low oxygen tension before cross- line repository was set up that can be interrogated on the linking in order to allow for an analysis of hypoxia-induced basis of user interest (http://shiny.cecad.uni-koeln. changes in the RNA-binding landscape. After 18 hours of in- de:3838/mIMCD_RBPome). cubation with 4-SU, crosslinking was performed in living cells using a UVA pulse and polyA-tailed RNAs were captured using Characterization of the Proteins Contained in the oligo-dT beads (Figure 1A). Visualization of proteins con- RBPome of mIMCD-3 Cells tained in small aliquots of these precipitates on a silver gel On the basis of these findings, we went on to perform a detailed confirmed efficient crosslinking (Figure 1B). The clear sepa- comparison of the proteins identified in our RIC with the ration of crosslinked and noncrosslinked samples is depicted compendium1—including both their list of mouse proteins in a principal component analysis (Figure 1C). After RNase as well as the mouse orthologs of the human RBPs. There treatment of the precipitates to remove the non–protein was a large overlap with the vast majority of the 1058 class bound RNA fraction, samples were analyzed for protein con- I/class II RBPs (Figure 2A). Interestingly, 25 of the proteins tent using mass spectrometry (Figure 1A). In total, we iden- had not been identified by previous screens using mouse or tified 1302 proteins (Supplemental Table). Because differences human cells, suggesting that these proteins may be specific in oxygen tension did not lead to major global changes (as RBPs in differentiated kidney epithelial cells (Figure 2A indicated in Figure 1C, Supplemental Figure 1), we decided and 1D, see also “RBPome” tab in the online repository). To to pool all samples irrespective of the treatment at this point to further characterize the enrichment of RBPs in our dataset com- allow for a comprehensive identification of RBPs. A detailed pared with the cellular proteome of mIMCD-3 cells, we per- comparative analysis of hypoxia-induced changes is provided formed quantitative label-free proteomics of whole-cell lysates. below. Because the comparison of protein abundance in cross- These studies identified 6033 individual proteins that were later linked over noncrosslinked samples is generally used as an used as a background for GO-term analyses. For quantification indicator of confidence that a protein identified by RIC is truly of hypoxia-associated changes in abundance this list was reduced an RBP, we classified the proteins according to the following to proteins identified in at least two of three replicates of either criteria (Supplemental Figure 2A). Class I RBPs are signifi- condition (normoxia or hypoxia), resulting in 4883 quantifiable cantly enriched (on the basis of iBAQ intensities) in the cross- proteins (see “Total proteome” tab in the online repository linked samples over noncrosslinked samples (t test, FDR,0.1; and Supplemental Table). The comparison of the mIMCD-3 s0=0.1). Furthermore, all proteins for which significance proteome with the list of known mouse RBPs listed in the could not be calculated due to lack of detection in noncros- compendium1 revealed that about 28% of these proteins were slinked samples (identification in #1 of 6 replicates), despite RNA-associated (Figure 2B). Performing the same comparison clear presence after crosslinking (identification in $4of6 for the class I RBPs or both class I and II RBPs from our RIC replicates), were attributed to class I. A total of 510 proteins experiment showed, as expected, a much higher proportion of fulfilled these criteria (Supplemental Table). However, known RNA binders (approximately 91%; Figure 2B).

analysis. Here, the ten most signiﬁcantly overrepresented (black) and underrepresented (gray) GO terms for “molecular function” and “biological process (slim)” are depicted. Statistical signiﬁcance was determined using the Fisher exact test (Perseus software; FDR,0.05). BP slim, biological process; GO, gene ontology; MF, molecular function.

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A Gene name Protein name (Uniprot and MGI) selection criteria Ctc1 Conserved telomere maintenance component 1/CTS telomere maintenance complex componen1t # Hars Histidyl-tRNA synthetase # Wasl Neural Wiskott-Aldrich syndrome protein # Tmem33 Transmembrane protein 33 # Mpdz multiple PDZ domain protein # Arhgap21 Rho GTPase activating protein 21 # Bclaf3 Bclaf1 and Thrap3 family member 3 # Ephx1 Epoxide hydrolase 1, microsomal # Gadd45gip1 Growth arrest and DNA-damage-inducible, gamma interacting protein 1 # No66 Bifunctional lysine-specific demethylase and histidyl-hydroxylase NO66/ribosomal oxygenase 1 # Mcm5 DNA replication licensing factor MCM5/ minichromosome maintenance complex component 5 # Nme1/2 Nucleoside diphosphate kinase/Nucleoside diphosphate kinase B # Cdk1 Cyclin-dependent kinase 1 # Ptrf Polymerase I and transcript release factor/RalBP1-associated Eps domain-containing protein 1 # Gm9242;Gm6793 Predicted pseudogene 9242; predicted gene 6793 # Usb1 U6 snRNA phosphodiesterase/U6 snRNA biogenesis 1 # Dhx37 DEAH (Asp-Glu-Ala-His) box polypeptide 37 # Mrpl47 Mitochondrial ribosomal protein L47 # Kif13b Kinesin-like protein/kinesin family member 13B #

Usp7 Ubiquitin carboxyl-terminal hydrolase 7/ubiquitin specific peptidase 7 * Ccdc94 Coiled-coil domain-containing protein 94/YJU2 splicing factor * Oasl2 2'-5' oligoadenylate synthetase-like protein 2 * Reps1 RalBP1 associated Eps domain containing protein 1 * Bcas2 Pre-mRNA-splicing factor SPF27/breast carcinoma amplified sequence 2 * Arid2 AT rich interactive domain 2 (ARID, RFX-like) *

B previously detected significant by RIC in t-test

Gene name Protein name Mm Hs Class Novel RBP +CL vs -CL

Mfap1a/b Microfibrillar-associated protein 1A/B Mfap1b MFAP1 class I +

Gadd45gip1 Growth arrest and DNA damage-inducible proteins-interacting protein 1 n.d. n.d. class I ++

Hic2 Hypermethylated in cancer 2 protein Hic2 n.d. class I +

CDMFAP1 GADD45GIP1 HIC2 MFAP1 GADD45GIP1 HIC2 Crosslinked ++-- +- GFP FLAG PNK-assay 75 40 85

Western blot 75 40 85

DAPI DAPI

merge merge

Figure 3. RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specificRBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (2CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on

570 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 564–576, 2019 www.jasn.org BASIC RESEARCH

This is not a general capacity of nucleic-acid binding but spe- RNA binders so far. Firstly, we visualized the subcellular lo- cific to RNA, which is illustrated by the overlap with proteins calization of these proteins. Mfap1a/b and Hic2 were predom- contained in the gene ontology (molecular function) terms inantly nuclear with only a slight cytoplasmic signal, whereas “RNA-binding” and “DNA-binding” (Figure 2C). Even Gadd45gip1 showed both a (peri)nuclear and cytoplasmic lo- though the number of proteins overlapping with the term calization (Figure 3C). In order to validate their RNA-binding “RNA-binding” was much larger, a significant number of pro- potential we employed PNK assays, revealing a clear-cut ra- teinswerealsoassumedtobindtoDNAandcouldbepartof dioactive band at the size of the respective protein with a the group of “dual binders” that have recently gained increas- strong enrichment in the crosslinked samples (Figure 3D). ing attention (Figure 2C).22 Using the whole-cell proteome These results did not only confirm Hic2 and Mfap1a/b for (described in Figure 2B) as a reference, we asked which known the first time on an individual protein level to be RNA binders protein domains were enriched among the identified RBPs. but also showed that our list of entirely novel mammalian Both PFAM and SMARTenrichment analyses clearly showed RBPs contains true RNA binders as indicated by the result nearly all of the most highly enriched domains to be classic for Gadd45gip1. RBDs, with helicase-associated domains on the one hand and the classic RBDs RRM and KH on the other hand being the Cilia-Associated RBPs most prominent terms (Figure 2D). Gene ontology as well as Primary cilia are a hallmark of differentiated renal tubular pathways analyses—as before, using the proteome as a refer- epithelial cells. To demonstrate ciliation upon serum starva- ence—of the 510 class I RBPs revealed that top terms in all tion–induced differentiation we stained for cilia markers. As three GO categories again demonstrated a highly significant shown in Figure 4A and Supplemental Figure 4A, our overrepresentation of RNA-associated processes (Figure 2E, mIMCD-3 cells showed a consistently high degree of cilia- Supplemental Figure 2, B and C). tion after 30 hours of serum starvation. We next asked whether RBPs identified in our experiment may also be Proteins Identified for the First Time as RNA Binders in cilia-associated proteins. To address this question, the RBP Differentiated mIMCD-3 Cells compendium1 as well as our dataset of RBPs was analyzed for As described above, 25 class I RBPs identified in our RIC have overlaps with both cilia-associated protein complexes as not been reported in previous screens of the mammalian characterized by the “SYSCILIA” consortium24 and the cil- RBPome (Figure 2A). Figure 3A provides a list of these novel iary membrane-associated proteome that we had deter- and potentially context-specificRBPs(“novel RBPs”). As this mined using the “APEX” technology.25 Both RBP datasets exclusive detection in mIMCD-3 cells may not only be a con- contained a subset of cilia-associated proteins (Figure 4B, sequence of context-specific RNA-binding capacity but could Supplemental Table). The scatterplot in Figure 4C shows also be explained by absence of protein expression in the cell cilia-associated proteins identified in the two screens plotted lines used in previous screens, we queried these proteins against the enrichment by crosslinking. Regarding the 25 using a freely accessible proteome database (“Proteo- RBPs identified for the firsttimeinmIMCD-3cells micsDB”).23 This analysis clearly showed that all of our novel (Figure 3A)—of which none overlapped with the SYSCILIA RBPs (apart from Oasl2 and Gm9242 for which no data are and APEX datasets—we performed a more detailed litera- available in “ProteomicsDB”) are expressed in numerous cell ture search and identified five additional proteins (Kif13b, lines, including at least one cell line used in previous RIC Mpdz, Nme1/2, Wasl, Cdk1) to have a known ciliary function screens,1 (Supplemental Figure 3A). We decided to confirm (Figure 4D).26–30 Because ciliary targeting may be context- RNA binding for three proteins from our screen (Figure 3B). specific and none of these proteins had been examined in Specifically, Gadd45gip1 is one of the entirely novel RBPs mIMCD-3 cells before, we confirmed ciliary localization in (Figure 3A), whereas Hic2 and Mfap1a/b had been identified this cell type for Mpdz and Kif13b by immunofluorescence in previous screens but had not been confirmed as individual as a proof of principle (Supplemental Figure 3B).

gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 mm. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (2) samples and the associated RNA was labeled by T4 PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.

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ABMm RBPs (n=1914) Mm RBPs (n=1914) vs APEX vs SysCilia anti-acetylated tubulin anti-pericentrin 93 20

1821 1894

mIMCDs RBPs (n=1058) mIMCDs RBPs (n=1058) vs APEX vs SysCilia 62 10

DAPI merged 996 1048

C D

7 7 Crnkl1 6 6

Rrp12 5 Mov10 5 RsI1d1 Srsf1 Eif4b Kif13b 4 4 Caprin1 G3bp2 3 3 Wasl p value +CL vs -CL p value Pabpc1 +CL vs -CL p value Mpdz 10 2 10 2 -log -log Cdk1 1 1

Nme1/2 0 0

–2 –1 0 1 2 3 4567 –2–101234567

log2 FC +CL vs -CL log2 FC +CL vs -CL

Figure 4. Cilia-associated proteins show RNA-binding capacity. (A) Immunofluorescence imaging of ciliated mIMCD-3 cells. The cells were serum starved for 30 hours to induce ciliogenesis, fixed, and stained with antibodies specific for actylated tubulin (green) and pericentrin (magenta). DAPI (blue) was used as a nuclear counterstain. Scale bar, 20 mm. (B) Comparison of known mouse RBPs1 and mIMCD-3 RBPs with cilia-associated proteins as described by Boldt et al.21 (2016) (SYSCILIA) and Kohli et al.22 (2017) (APEX). The compendium of known mouse RBPs (n=1914) contains 93 ciliary proteins as determined by APEX (red) and 20 ciliary proteins characterized by the SYSCILIA consortium (blue). The mIMCD-3 RBPome (n=1058) shares 62 and ten ciliary proteins with the APEX and the SYSCILIA datasets, respectively. (C) Identity of ciliary proteins in the mIMCD-3 RBPome. Depicted in red are proteins overlapping with the APEX dataset22 and/or the SYSCILIA dataset.21 For proteins significantly enriched in the mIMCD-3 RBPome the protein name is indicated. Gray, mIMCD-3 proteins without correspondence to APEX and SYSCILIA datasets. For details of the scatter plot refer to Figure 1D. (D) Scatter plot illustrating mIMCD-3 RBPs (never identified in mammalian RIC experiments before) associated with a known ciliary function. A PubMed-based literature search was performed for the 25 mIMCD-3 RBPs (black) shown in Figure 3A. Protein names are indicated for RBPs associated with the search term “cilia.” For details regarding the scatter plot refer to Figure 1D. +CL, crosslinked; 2CL, not crosslinked; FC, fold change; vs, versus.

Modulation of the RBPome and Proteome by Hypoxia dynamic changes of RBP–target RNA binding, rather than Because hypoxia plays a critical role in renal (patho)physiology hypoxia-associated changes in abundance of the respective and is known to affect mRNA stability and translation, we RBPs, we used a limited duration of hypoxia (6 hours). This addressed the question of how hypoxia might affect the in- treatment did not affect ciliation (Supplemental Figure 4A) teraction between RBPs and their target mRNAs. As indicated but led to a pronounced stabilization of Hif1a and its enrich- above, 50% of the cells were exposed to deﬁned levels of hyp- ment in the nucleus (Supplemental Figure 4, B and C), indi- oxia before crosslinking (Figure 1A). To obtain a view on cating activation of the hypoxia transcriptional program.

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5 ABP4ha2 4.5

4 6 +CL vs 6 -CL Kdm3a 3.5 (n=455)

3 Ndrg1 Plod2 142 113 P4ha1 2.5 Slc2a1 Normoxia p value 170 10 (n=289) 6 30 2 -log 6 Hypoxia 1.5 (n=206) 1

0.5

-1 -0.5 0 0.5 1 1.5 2

log2 FC hypoxia vs normoxia

C D 5

7 4.5 6 4 Mrps30 5 Hic2 Casc3 D2Wsu81e Ddx1 3.5 4 Xab2 Ppan Dhx57 Rps15 Mcat Cdc5l 3 3 Ppp1r10 2 Fbll1 2.5 p value 10 1 Rbmx 2

Cnot1 -log 0 Sod1 Eif3l Tmem33 1.5 -1 Rplp2

FC Normoxia +CL vs -CL 1 2 -2 Nme1/2

log Anxa2 0.5 Sod1 -3 Rplp2 Tcp1 Rps4x Sdad1 0 Nme1/2 Tmem33 -3 -2 -1 0 1 234 5 6 7 Mcat Ppp1r10 log2 FC Hypoxia +CL vs Hypoxia -CL -1 -0.5 0 0.5 1 1.5 2

log2 FC hypoxia vs normoxia

E Hentze et al., 2018 Significant in t-test RBPomes Hypoxia Normoxia novel Mm Hs Class Gene name Protein name +CL vs -CL +CL vs -CL RBP Nme 1/2 Nucleoside diphosphate kinase;Nucleoside diphosphate kinase B++ class I Dhx57 Putative ATP-dependent RNA helicase DHX57+ ++ class I Mcat Malonyl-CoA-acyl carrier protein transacylase, mitochondrial+ + + class I Ppp1r10 Serine/threonine-protein phosphatase 1 regulatory subunit 10+ ++ class I Rplp2 60S acidic ribosomal protein P2 + + + class I Sod1 Superoxide dismutase [Cu-Zn] ++class I Tmem33 Transmembrane protein 33 + class I +

Figure 5. The RBPome is modulated by oxygen tension. (A) Volcano plot illustrating differentially abundant proteins between the proteomes of hypoxia-treated and normoxic mIMCD-3 cells. The 2log10 P value is plotted against the log2 fold change (hypoxia versus normoxia). Signiﬁcantly regulated proteins are above the cutoff line and are indicated by name (Perseus software, t test, FDR,0.1, s0=0.1). In total, 4883 proteins were plotted. Red, known Hif1a targets30,31,34; blue, Kdm3a, manually curated from the literature as an Hif1a target.30 Average fold change of total number of proteins (n=4883) 0.015; average fold change of Hif1a targets (n=94) 0.45. (B) Venn diagram depicting the comparison of RBPomes of mIMCD-3 cells grown in hypoxic and normoxic conditions. Of the 289 (normoxia) and 206 (hypoxia) RBPs reaching the threshold of class I RBPs, six and six proteins are exclusively associated with normoxia

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In order to check what effect this treatment had on global crosslinking in normoxic and hypoxic cells and found a set protein expression, we performed quantitative label-free proof proteins to be more strongly enriched in one of the con- teomics (Figure 5A, see also “Hypoxia versus Normoxia” tab ditions (normoxia 41 proteins, hypoxia 41 proteins; Supple- in the online repository). No proteins were downregulated mental Table, see also “Hypoxia versus Normoxia” tab in the and the six upregulated proteins reaching significance online repository). Using this approach, seven of the 12 pro- (FDR,0.1, s0=0.1) had all been previously reported as hyp- teins (Figure 5B) identified to be specific to one of the two oxia-signaling associated.10,11,31–34 Additionally, by calculat- conditions were also found to be outside of the 95% pre- ing the average fold change of the total number of proteins in diction interval, increasing the confidence in these candi- the whole dataset (FC=0.015, n=4883) and the average fold dates (indicated in black, Figure 5C). Importantly, none change of all Hif1a targets (FC=0.45, n=94) depicted in Figure of the 12 proteins was altered significantly between the 5A, we confirmed a trend toward a positive regulation of all proteomes (Figure 5D), clearly pointing toward hypoxia- Hif1a targets in the hypoxia-treated samples. Moreover, our associated differential binding. Figure 5E provides more dataset revealed that 46 proteins were exclusively detected information on the seven proteins identified by both ap- under hypoxic conditions but were never identified in nor- proaches (i.e., derived from the overlap of Figure 5B and moxia, including Hif1a (Supplemental Table). Seventeen pro- C). All putatively hypoxia-associated RBPs in our screen teins were never measured in hypoxia, including the RNase had previously been associated with oxygen metabolism Drosha that is known to be downregulated in hypoxic conditions35 and signaling in the literature (Supplemental Table). Two (Supplemental Table). Taken together, as intended, 6 hours of them are among the novel RBPs identified in our study, of hypoxia only had a limited effect on global protein ex- Nme1/2 and Tmem33, both of which reach significance only pression with few proteins being regulated significantly (Fig- in hypoxic samples (Figure 5E). ure 5A, see also “Hypoxia versus Normoxia” tab in the online repository and Supplemental Figure 4D). We then went on to compare the RBPome of cells grown under different oxygen DISCUSSION tension. To this end, we performed separate t tests for the normoxia and hypoxia datasets (3 +CL versus 3 2CL; FDR Here, we report the first global analysis of RBPs in ciliated 0.1, S0=0.1) and compared these with the t test of the whole epithelial kidney cells, paving the road toward kidney re- dataset (6 +CL versus 6 2CL) shown in Figure 1D (Figure search gaining an insight into the rapidly evolving field of 5B). This revealed six proteins (Nme1/2, Anxa2, Rplp2, RNA-proteininteractionsincellularandorganbiology.Our Sdad1, Sod1, Tmem33) that exclusively reached the FDR approach (ciliated kidney cells in hypoxia and normoxia) did threshold of class I RBPs in hypoxic cells, and this was also not only yield an atlas of 1058 RBPs but also allowed for the the case for six proteins (Dhx57, Mcat, Ppp1r10, Rps4x, Se- identificationof25novelRBPsthatwere notdetectedinother cisbp2l, Tcp1) in normoxic cells (Figure 5B, see also systems before. The fact that these novel RBPs are expressed “Hypoxia versus Normoxia” tab in the online repository in other cultured cell lines that had been used for RIC screens and Supplemental Table). To allow for a more quantitative previously emphasizes the importance of studying such in- analysis we next correlated the enrichment reached by teractions in various cell types and conditions. However, it

or hypoxia, respectively. Gray, normoxia-associated RBPs (class I RBPs reaching statistical significance when comparing three +CL versus three 2CL samples in normoxia); blue, hypoxia-associated RBPs (class I RBPs reaching statistical significance when comparing three +CL versus three 2CL samples in hypoxia); red, six +CL versus six 2CL samples (455 class I RBPs reaching statistical significance in the comparison of the total dataset comprising six +CL and six 2CL samples). (C) Scatter plot showing the correlation of hypoxia log2 fold changes (+CL versus 2CL) on the x axis versus the log2 fold change values of normoxia (+CL versus 2CL) on the y axis. The linear regression was calculated with R (black line, lm () method, formula: y=0.3170+0.7069*x). RBPs beyond the calculated 95% prediction interval (outside the gray lines) show a significantly different FC in the two conditions, sometimes going in opposite directions (from positive to negative), suggesting a regulation of the binding to target RNAs in normoxia or hypoxia. Red, class I and II RBPs (with names for proteins above and below the calculated prediction interval); big circles, significant in t test (FDR,0.1); small circles, NS in t test (FDR$0.1); black, seven RBPs below and above the calculated prediction interval matching the 12 differentially bound RBPs in Figure 5B; gray, others. (D) Volcano plot illustrating the abundance of differentially bound RBPs (Figure 5B) between the proteomes of hypoxia-treated and normoxic mIMCD-3 cells. Red, differentially bound RBPs significant in normoxia; blue, differentially bound RBPs significant in hypoxia. Two proteins (Dhx57and Secisbp2l) were not quantified in the proteome. For details regarding the volcano plot refer to Figure 5A. (E) List of high- confidence RBPs associated with either hypoxia or normoxia. This table shows the seven of 12 differentially bound RBPs that were measured in hypoxia- or normoxia-treated cells, respectively, and in addition were beyond the calculated 95% prediction interval. Given are gene and protein names, significance in t test (+CL versus 2CL, in hypoxia or normoxia), information on the presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium,1 classification in the mIMCD-3 RBPome (class), and classification as to whether the protein is a novel RBP. +CL, crosslinked; 2CL, not crosslinked; FC, fold change; vs, versus.

574 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 564–576, 2019 www.jasn.org BASIC RESEARCH has to be noted that, apart from context-specificchangesin ACKNOWLEDGMENTS RNA-binding capacity, technical differences (e.g.,astoMS/ MS) can contribute to such a finding. We thank Serena Greco-Torres for excellent technical assistance. Although a few studies have already pointed toward a This work was supported by the Nachwuchsgruppen NRW crucial role of specific RBPs regarding kidney physiology,6 program of the Ministry of Science North Rhine Westfalia (MIWF, the role of this class of proteins in ciliary biology is entirely to R.-U.M.), the German Research Foundation (DFG; MU3629/2-1 unclear. It is easy to believe that RBPs—which affect most to R.-U.M., BE2212 and KFO329 to T.B., SCHE1562/6 to B.S.), the biologic processes—may also affect cilia formation, signal- University of Cologne (Köln Fortune Program [139/2013 and 220/ ing, and breakdown through their profound effect on cellu- 2015] to K.H.), and a Fellowship by Boehringer Ingelheim Fonds lar RNA biology. However, it is even more intriguing that (to R.K.). C.D. acknowledges funding by the Klaus Tschira Stiftung datasets like the one presented here allow for the speculation gGmbH.ThemAbE7developedbyM.McCutcheonandS.Carroll —because a number of the RBPs detected are actually pro- was obtained from the Developmental Studies Hybridoma Bank, teins that have been shown to localize to primary cilia—that created by the National Institute of Child Health and Human the cilium might be a novel site of action for RBPs. It is Development of the National Institutes of Health, and maintained important to note that most of these proteins do not neces- at The University of Iowa, Department of Biology, Iowa City, sarily only localize in cilia and also have other cellular func- IA 52242. tions. Consequently, such a hypothesis will require further F.F. and R.-U.M. designed the study; M.I., C.R., R.K., M.K., R.E., studies comparing ciliated to nonciliated cells and address- I.A., K.H., C.K.F., X.L., M.P., and F.F. performed experiments; C.D., ing the potential mode of action of putative ciliary RBPs. M.M.R., C.R., R.K., K.B., and F.F. analyzed the data; F.F., M.I., R.K., It will now be extremely interesting to move these efforts and R.-U.M. prepared the figures; R.-U.M., M.I., R.K., F.F., C.K.F., from a characterization of the RBPome toward function in B.S., and T.B. drafted and revised the paper; all authors approved the kidney (patho)physiology. Here, in order to provide some final version of the manuscript. first clues, we focused on the potential effect of hypoxia taking into account that the renal medulla is a site of limited oxygen tension and that lack of oxygen is one of the key DISCLOSURES factors in both AKI and CKD.36 A very interesting example None. of the potential role of RNA-protein interaction in the response to hypoxia is the targeting of specificmRNAsto polysomes for translation via the 59 cap binding of the SUPPLEMENTAL MATERIAL HIF2a-RBM4-eIF4E2 complex.37,38 Furthermore, key as- pects regarding hypoxia are cellular metabolic adaptations This article contains the following supplemental material online at (e.g., activation of glycolysis) and recent work has suggested http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018090914/-/ that a large number of metabolic enzymes possess RNA- DCSupplemental. binding capacity.2,3 Interestingly, a significant number of Table of contents—Supplemental Tables and Figures. enzymes involved primarily in glucose handling in the kid- Supplemental Figure 1. Heatmap of 4 replicates of RNA inter- ney were identified as RBPs either by our RIC screen or actome capture experiments in mIMCD-3 cells. previously published data3 (Supplemental Figure 5A). Im- Supplemental Figure 2. Characterization of the mIMCD-3 RNA portantly, two of the six proteins depicted in Supplemental interactome. Figure 5A—Aldolase a and Enolase 1—arebelowthe95% Supplemental Figure 3. Expression and ciliary localization of novel prediction interval, suggesting significantly increased RNA RBPs. binding in hypoxic conditions (Supplemental Figure 5, Supplemental Figure 4. Hypoxia signaling–associated changes. B and C). Future experiments identifying the RNA mole- Supplemental Figure 5. RNA-binding proteins in hypoxia-induced cules bound by these enzymes and their effect on enzyme metabolic changes. activity will help understanding the effect of this additional Supplemental Table. mIMCD-3 RNA interactome and proteome. layer of regulation regarding hypoxia-induced alterations of cellular metabolism. In summary, our study provides the first global view on REFERENCES RBPs in ciliated kidney epithelial cells and will—by providing a visualization of large-scale datasets on both the whole-cell 1. Hentze MW, Castello A, Schwarzl T, Preiss T: A brave new world of RNA- proteome and the RBPome of mIMCD-3 cells—serve as a re- binding proteins. Nat Rev Mol Cell Biol 19: 327–341, 2018 source for future targeted analyses of specificRBPsandthein- 2. Liao Y, Castello A, Fischer B, Leicht S, Föehr S, Frese CK, et al.: The teractionwith their targets in kidney (patho)physiology. In order cardiomyocyte RNA-binding proteome: Links to intermediary metabolism and heart disease. 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