Abstract: TH-PO232

Identification of the RNA Interactome of Ciliated Tubular Cells and Its Modulation by Hypoxia

Session Information

Category: Acute Kidney Injury

  • 001 AKI: Basic

Authors

  • Mueller, Roman-Ulrich, Renal Unit, University Hospital Cologne, Koeln, Germany
  • Kaiser, Rainer, Renal Unit, University Hospital Cologne, Koeln, Germany
  • Rill, Constantin, Renal Unit, University Hospital Cologne, Koeln, Germany
  • Fabretti, Francesca, Renal Unit, University Hospital Cologne, Koeln, Germany
  • Ignarski, Michael, Renal Unit, University Hospital Cologne, Koeln, Germany
  • Rinschen, Markus M., Renal Unit, University Hospital Cologne, Koeln, Germany
  • Atanassov, Ilian, Max Planck Institute for Biology of Aging, Cologne, Germany
  • Schermer, Bernhard, Renal Unit, University Hospital Cologne, Koeln, Germany
  • Benzing, Thomas, Renal Unit, University Hospital Cologne, Koeln, Germany
Background

RNA-binding proteins (RBPs) control the fate of all RNA species and every step of messenger RNA generation and processing. Through specific binding of their targets, RBPs heavily influence the cellular transcriptome and proteome. The scale of their impact has been shown by recent studies that have linked RBPs to a number of human pathologies, ranging from neurological disorders to tumor growth. The global effect of RBPs in kidney physiology has never been assessed until now. Here, we identify RNA binding proteins in renal epithelial cells under both hypoxic and normoxic growth conditions as well as candidate RBPs that may play key roles in hypoxia signaling of of the kidney.

Methods

Using an oligo(dT) capture approach to precipitate mRNA-protein complexes in ciliated mIMCD-3 cells, we aimed to identify the kidney-specific mRNA interactome using mass spectrometry. UV-crosslinked samples were either exposed to hypoxia (1% O2) or grown under normoxic conditions. In parallel, the proteome of whole cell lysates was identified to assess the total amount of protein in comparison to RBPs detected under different conditions. Using TALEN-mediated genome engineering for the creation of transgenic human cell lines, specific candidates were validated as RNA binding protein with the Polynukleotide (PNK) assay. Subsequent immunofluorescence studies on the same cell lines revealed the localization pattern of these RBPs.

Results

Our data revealed over 350 significant mRNA interactors and more than 300 additional candidate RBPs, 84 of which have not been described as RNA binding proteins in common data bases. We define these proteins as the renal epithelial cell mRNA interactome. Based on whole cell proteome analyses we hypothesize that the increased detection of RBP candidates in the hypoxia-treated Oligo(dT) captured samples is indeed due to differential binding to their target transcripts.

Conclusion

RNA binding proteins are important players in general cell biology and may also play a key role in the cellular response to hypoxia of kidney cells. Our data identify the first set of RBPs specific to renal epithelial cells and aid to understand their role in both kidney physiology and pathology, adding another regulatory layer to the diverse biology of the kidney.

Funding

  • Government Support - Non-U.S.