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Kidney Week

Abstract: FR-PO082

AATF/Che-1 Controls Ribosomal Biogenesis Through Direct Binding of Ribosomal RNA and R-Protein Encoding mRNAs

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Kaiser, Rainer, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany
  • Ignarski, Michael, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany
  • Rill, Constantin, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany
  • Van nostrand, Eric, UCSD , La Jolla, California, United States
  • Rinschen, Markus M., Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany
  • Schermer, Bernhard, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany
  • Benzing, Thomas, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany
  • Hoepker, Katja, University of Cologne, Köln, Germany
  • Fabretti, Francesca, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany
  • Mueller, Roman-Ulrich, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany
Background


AATF/Che-1 (Apoptosis Antagonizing Transcription Factor) is an RNA Pol II binding transcription factor that has been shown to play crucial roles in multiple essential cellular pathways - e.g. DNA damage response and mTOR signaling. It further has been linked to acute kidney injury. However, the exact molecular function of this protein is not known.

Methods

We have identified AATF as a putative RNA binding protein (RBP) in murine kidney cells. Here, we use Enhanced Crosslinking and Immunoprecipitation (eCLIP) as well as RNA editing followed by sequencing (TRIBE) techniques to identify both coding and non-coding RNAs bound by AATF. Binding of RNA as well as validation of specific targets was done by PNK assays and RIP-qPCR. Using TALEN-mediated genome engineering, we have created a set of transgenic cell lines that express GFP-tagged WT AATF or AATF mutant proteins. In addition, we perform MS/MS studies to uncover proteins interacting with AATF.

Results


Here we validate AATF as an RNA binding protein and identify hundreds of RNA targets bound by AATF. Analysis of transcripts bound by AATF were enriched for ribosomal and other non-coding RNA biotypes. More specifically, the 45S rRNA precursor was one of the most enriched transcripts bound to AATF. Moreover, both structural constituents of the RNP and components of the ribosome biogenesis machinery were strongly overrepresented among the mRNA transcripts.
Both the 45S pre-ribosomal precursor and its product, 18S rRNA, were depleted upon knockdown of AATF. However, AATF did not only interact with RNA molecules associated with ribosomal function and integrity, but also with a large number of protein components of the rRNP and other RNA binding proteins.

Conclusion


Our study validates AATF as an RNA binding protein and reveals yet another link between rRNA metabolism, nucleolar integrity and other essential pathways such as the DNA damage response. An impact on ribosome abundance and functionality mediated by RNA binding could be an important feature of the role of AATF in carcinogenesis and may open new ways to address its role as a potential therapeutic target. To this end, a better understanding of its molecular function is of great importance.

Funding

  • Private Foundation Support