1. Research Background:

Kidney disease is a very common aging-associated disorder and is a major risk factor for cardiovascular morbidity and mortality1. We study the molecular mechanisms involved in the pathogenesis of renal function decline and aim to identify novel strategies to prevent kidney disease. In this context, RNA-biology is the central focus of our work. Despite the known impact of both coding and non-coding RNA molecules and their metabolism as well as their interaction with other biomolecules such as DNA and proteins little is known about their impact on aging and kidney disease. Consequently, this research harbors a great potential to identify novel pathomechanisms and therapeutic targets.

2. Research questions addresses by the group:

  • How do long non-coding RNAs modulate aging-associated phenotypes and renal function decline? Is this effect dependent on cell-type specific expression and functions?
  • Which RNA-protein binding events are altered by kidney disease and can be therapeutically targeted?
  • How does RNA binding influence the molecular function of non-classical RNA binding proteins (enigmRBPs)? Is this change in molecular function involved in aging-associated disease?
  • Can RNA-protein binding be altered to protect from kidney injury?
  • Does aging and kidney disease influence RNA modifications? How do these RNA modifications impact RNA function and binding to other biomolecules? Are these modifications involved in the pathogenesis of aging-associated disease?

3. Possible projects:

 

  • Project 1: The impact of RNA modifications and their modulation on aging-related phenotypes in cellular biology
    Advances in the understanding of the chemical labelling of DNA has granted us access to a unique field of study: epigenetics. This has opened up a whole new level of information, helping us understand how and why life behaves and reacts in the varied fashion we observe. However, DNA represents only one side of the coin. The chemical labeling of RNA on the other hand is largely unexplored. Harnessing the capabilities of third-generation direct RNA sequencing, our project aims to shed some light on the field of epitranscriptomics2. While recent improvements in the detection and mapping of RNA modifications have led to epitranscriptomics being elected “method of the year 2016”, functional studies of the epitranscriptome lag behind those of the epigenome due to a lack of robust sequencing technologies. This project sets out to exploit the great potential of nanopore sequencing technology in direct RNA sequencing for the identification of site-specific RNA modifications. Specifically, we will use this technology to elucidate aging-associated alterations in the RNA modification landscape and obtain an insight into their relevance in human disease.
  • Project 2: Elucidating the role of lncRNAs in acute kidney injury
    Using RNAseq datasets of the kidney after cisplatin3 and ischemia-reperfusion induced acute kidney injury in a mouse model combined with datasets on mice pre-treated by caloric restriction, hypoxic preconditioning and prolyl hydroxylase inhibitors we were able to identify a number of known and novel lncRNAs to be significantly dysregulated as candidates to be involved in the protection from acute kidney injury. Based on this knowledge, we have designed the strategies for the CRISPR-Cas9 based generation of 5 lncRNA knockout mouse lines that are currently generated in the CECAD transgenesis facility. These mouse lines will now be analyzed focusing on renal phenotypes including susceptibility to AKI and preconditioning-mediated resistance. The phenotypic analyses will be coupled with molecular biology to unravel the entirely unknown molecular function of these lncRNAs employing state-of-the-art methods such as CHART-MS to identify protein, RNA and DNA interactors. Furthermore, the spatial resolution of their expression in the kidney will be taken to the cellular level using kidney scRNAseq4.

4. Applied Methods and model organisms:

  • model organisms: cell culture5,6, C. elegans7–9 and mouse3,10
  • all standard methods in molecular and cellular biology (such as molecular cloning, Western Blotting, Immunofluorescence etc.)
  • state-of-the-art methods regarding RNA-RNA and RNA-protein interactions, e.g. CLIP (crosslinking and immunoprecipitation)5–7, CHART-MS, RNA interactome capture, proteomics3, RNA-sequencing (including single-cell RNAseq)4,10, RNA modifications11
  • Direct RNA sequencing using Nanopore technology
  • transgenesis and CRISPR-Cas9 mediated genome editing in mouse and nematode
  • bioinformatics analysis of the datasets obtained

5. Desirable skills and qualifications:

  • good training in and understanding of standard techniques in molecular and cellular biology
  • interest in tackling clinically relevant questions using these techniques in cell culture and model organisms
  • FELASA certificate would be desirable, but can also be obtained the project
  • basic bioinformatics skills would be desirable, but can also be obtained during the first months of the project. However, an interest in the analysis of large-scale datasets under the supervision of a bioinformatician is required

6. References:

  1. 1. Hsu, R. K., McCulloch, C. E., Dudley, R. A., Lo, L. J. & Hsu, C. Temporal changes in incidence of dialysis-requiring AKI. J. Am. Soc. Nephrol. JASN24, 37–42 (2013).
  2. Saletore, Y. et al. The birth of the Epitranscriptome: deciphering the function of RNA modifications. Genome Biol.13, 175 (2012).
  3. Späth, M. R. et al. The proteome microenvironment determines the protective effect of preconditioning in cisplatin-induced acute kidney injury. Kidney Int. 95, 333–349 (2019).
  4. Karaiskos, N. et al. A Single-Cell Transcriptome Atlas of the Mouse Glomerulus. J. Am. Soc. Nephrol. JASN 29, 2060–2068 (2018).
  5. Ignarski, M. et al. The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells. J. Am. Soc. Nephrol. JASN 30, 564–576 (2019).
  6. Kaiser, R. W. J. et al. A protein-RNA interaction atlas of the ribosome biogenesis factor AATF. Sci. Rep. 9, 11071 (2019).
  7. Esmaillie, R. et al. Activation of Hypoxia-Inducible Factor Signaling Modulates the RNA Protein Interactome in Caenorhabditis elegans. iScience 22, 466–476 (2019).
  8. Gharbi, H. et al. Loss of the Birt-Hogg-Dubé gene product folliculin induces longevity in a hypoxia-inducible factor-dependent manner. Aging Cell 12, 593–603 (2013).
  9. Müller, R.-U. et al. The von Hippel Lindau tumor suppressor limits longevity. J. Am. Soc. Nephrol. JASN 20, 2513–2517 (2009).
  10. Johnsen, M. et al. The Integrated RNA Landscape of Renal Preconditioning against Ischemia-Reperfusion Injury. J. Am. Soc. Nephrol. JASN 31, 716–730 (2020).
  11. Dal Magro, C. et al. A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure. Angew. Chem. Int. Ed Engl. 57, 7893–7897 (2018).