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The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells

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The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells BASIC RESEARCH www.jasn.org The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells 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 quantita- tive 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 pro- vide 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] Published online ahead of print. Publication date available at Copyright © 2019 by the American Society of Nephrology www.jasn.org. 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 differenti- ated 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 plas- mids 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.
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