1. Research Background:

Kidney disease is a very common aging-associated disorder and is a major risk factor for cardiovascular morbidity and mortality. We study the molecular mechanisms involved in the pathogenesis of renal function decline and aim to identify novel strategies to prevent and/or cure kidney disease. In this context, modulation of cellular metabolism as a strategy to increase stress resistance and treat kidney disease is the central focus of our work. Considering both, the growing body of evidence regarding the impact of RNA-binding proteins in metabolic processes and the lack of knowledge regarding non-coding RNAs and RNA-protein interactions in kidney disease, we have a special interest in RNA-biology. Consequently, this research harbors a great potential to identify novel pathomechanisms and therapeutic targets. Importantly, all basic research in our lab is accompanied by clinical trials translating the findings from bench to bedside.

2. Research questions addressed by the group:

• How do interventions that mediate longevity and stress resistance modulate cellular metabolism?
• Which metabolic pathways are crucial to organ protection? Which molecules are involved and how do they mediate their effect?
• How do interactions between RNA and RNA-binding proteins influence cellular metabolism?
• Which RNA-protein binding events are altered in kidney disease?
• How do RNA-binding proteins modulate aging-associated phenotypes and renal function decline? Is this effect dependent on cell-type specific expression and functions?
• Can RNA-protein binding be targeted therapeutically to protect from kidney injury?

3. Possible project:

Riboregulation of the aldolase family in kidney disease and nephroprotection

This project is based on the recent observation that Sorbitol-dehydrogenase SORD, a well-described enzyme of the polyol pathway, has the capacity to bind RNA. As presented in our preliminary data, SORD binds RNA differentially upon IRI. We therefore hypothesize that SORD is subject to riboregulation resultingin adaptation of the polyol pathway in AKI. Considering the importance of metabolic processes for the pathogenesis of AKI we structure our approach to achieve three major aims:

1. Identification of RNA-binding regions and characterization of transcripts interacting with SORD.
2. Analysis of the impact of RNA-binding on the enzymatic activity of SORD and its effects on the polyol pathway (riboregulation).
3. Unraveling the role of riboregulatory effects of SORD in vivo using nematode models of stress resistance and mouse models of AKI.

We expect that this project will significantly enhance the understanding of riboregulatory processes in metabolic pathways and shed light on new entry points for pharmacological treatment.

A novel m6A reader in autosomal dominant polycystic kidney disease

This project is based on our observation that an unpublished RBP – know to function as an m⁶-methyladenine (m⁶A) reader – shows increased association with RNA in kidneys of a polycystic kidney disease mouse model (Pkd1^RC/RC). Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of kidney failure affecting more than 7 million people worldwide. Therapeutic optionsare limited, consequently, improving our understanding of the molecular pathogenesis is crucial. However, how this RBP modulates disease onset and progression is unclear and its molecular interactors have not been elucidated. Based on the recent findings showing that m⁶A RNA methylation is an important driver of cystogenesis in several PKD models it is both intriguing and warranted to speculate that the new RBP is the central m6A reader in ADPKD. We, therefore, hypothesize that this RBP has a pro- cystogenic function in ADPKD and mediates the effects of the m⁶A RNA modification. Consequently, we structure our approach to achieve three major aims:

1. Identification of transcripts interacting with the identified RBP comparing the wildtype to the Pkd1 loss-of-function setting.
2. Analysis of the impact of m⁶A RNA methylation on the mRNA interaction partners of this RBP.
3. Examine in vivo modulation of adenine m⁶ methylation as a therapeutic strategy for ADPKD.

We expect that this project will significantly enhance the understanding of RNA- protein interaction and its modulation by the m6A RNA modification in the pathogenesis of ADPKD and shed light on new entry points for pharmacological treatment.

4. Applied Methods and model organisms:

• Model organisms: cell culture, C. elegans and mouse (including models of kidney disease)
• Standard methods in molecular and cellular biology
• State-of-the-art methods regarding proteomics, RNA-sequencing (including single- cell RNAseq), RNA-protein interactions, e.g. CLIPseq (crosslinking and immunoprecipitation), RIC (RNA-interactome capture)
• Transgenesis and CRISPR-Cas9 mediated genome editing
• 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 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 and key publications:

• Cukoski, S., Lindemann, C.H., Arjune, S., Todorova, P., Brecht, T., Kühn, A., Oehm, S., Strubl, S., Becker, I., Kämmerer, U., Torres, J.A., Meyer, F., Schömig, T., Hokamp, N.G., Siedek, F., Gottschalk, I., Benzing, T., Schmidt, J., Antczak, P., Weimbs, T., Grundmann, F., Müller, R.-U., 2023. Feasibility and impact of ketogenic dietary interventions in polycystic kidney disease: KETO-ADPKD—a randomized controlled trial. CR Med 4. doi.org/10.1016/j.xcrm.2023.101283
• Seufert L, Benzing T, Ignarski M, Müller RU. RNA-binding proteins and their role in kidney disease. Nat Rev Nephrol. 2022 Mar;18(3):153-170. doi: 10.1038/s41581- 021-00497-1.
• Ignarski M, Rill C, Kaiser RWJ, Kaldirim M, Neuhaus R, Esmaillie R, Li X, Klein C, Bohl K, Petersen M, Frese CK, Höhne M, Atanassov I, Rinschen MM, Höpker K, Schermer B, Benzing T, Dieterich C, Fabretti F, Müller RU. The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells. J Am Soc Nephrol. 2019 Apr;30(4):564-576. doi: 10.1681/ASN.2018090914.
• Kaiser RWJ, Ignarski M, Van Nostrand EL, Frese CK, Jain M, Cukoski S, Heinen H, Schaechter M, Seufert L, Bunte K, Frommolt P, Keller P, Helm M, Bohl K, Höhne M, Schermer B, Benzing T, Höpker K, Dieterich C, Yeo GW, Müller RU, Fabretti F. A protein-RNA interaction atlas of the ribosome biogenesis factor AATF. Sci Rep. 2019 Jul 30;9(1):11071. doi: 10.1038/s41598-019-47552-3.
• Esmaillie R, Ignarski M, Bohl K, Krüger T, Ahmad D, Seufert L, Schermer B, Benzing T, Müller RU, Fabretti F. Activation of Hypoxia-Inducible Factor Signaling Modulates the RNA Protein Interactome in Caenorhabditis elegans. iScience. 2019 Dec 20;22:466-476. doi: 10.1016/j.isci.2019.11.039.
• Johnsen, M., Kubacki, T., Yeroslaviz, A., Späth, M.R., Mörsdorf, J., Göbel, H., Bohl, K., Ignarski, M., Meharg, C., Habermann, B., Altmüller, J., Beyer, A., Benzing, T., Schermer, B., Burst, V., Müller, R.-U.,2020. The Integrated RNA Landscape of Renal Preconditioning against Ischemia-Reperfusion Injury. J. Am. Soc. Nephrol. 31, 716– 730. doi.org/10.1681/ASN.2019050534
• Späth, M.R., Bartram, M.P., Palacio-Escat, N., Hoyer, K.J.R., Debes, C., Demir, F., Schroeter, C.B., Mandel, A.M., Grundmann, F., Ciarimboli, G., Beyer, A., Kizhakkedathu, J.N., Brodesser, S., Göbel, H., Becker, J.U., Benzing, T., Schermer, B., Höhne, M., Burst, V., Saez-Rodriguez, J., Huesgen, P.F., Müller,R.-U., Rinschen, M.M., 2019. The proteome microenvironment determines the protective effect of preconditioning in cisplatin-induced acute kidney injury. Kidney Int. 95, 333–349. doi.org/10.1016/j.kint.2018.08.037
• Huppertz I, Perez-Perri JI, Mantas P, Sekaran T, Schwarzl T, Russo F, Ferring-Appel D, Koskova Z, Dimitrova-Paternoga L, Kafkia E, Hennig J, Neveu PA, Patil K, Hentze MW. Riboregulation of Enolase 1 activity controls glycolysis and embryonic stem cell differentiation. Mol Cell. 2022 Jul 21;82(14):2666-2680.e11. doi: 10.1016/j.molcel.2022.05.019.
• Ramalingam, H., Kashyap, S., Cobo-Stark, P., Flaten, A., Chang, C.-M., Hajarnis, S., Hein, K.Z., Lika, J.,Warner, G.M., Espindola-Netto, J.M., Kumar, A., Kanchwala, M., Xing, C., Chini, E.N., Patel, V., 2021. A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease. Cell Metabolism 33, 1234-1247.e7. doi.org/10.1016/j.cmet.2021.03.024