In Vivo Characterization of RNA-Targeting Small Molecule Drugs with Enhanced UV-Crosslinking Proteomics

• Applicant: Dr. Jakob Trendel
• Subject Area: Biochemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 492625837

Protein-RNA interactions are fundamental to a great number of biological processes and their manipulation has been a continuous pursuit in the effort to cure disease. Only recently the splicing modifier risdiplam became the first RNA-targeting small molecule drug to be approved by the FDA and EMA for the treatment of spinal muscular atrophy (SMA). Although targeting the central U1 complex of the spliceosome, risdiplam occurs to be specific in altering splicing of only the SMN2 gene and very few other off-target genes. It has therefore been speculated that additional, unknown factors might contribute to the specificity of the drug. In the past, we have developed XRNAX, an approach for UV-crosslinking proteomics, which allows for the quantification of protein residing on RNA in a system-wide fashion. We have now extended this approach and constructed UV-crosslinking devices based on light emitting diodes (LEDs), which in our preliminary experiments strongly improve crosslinking of protein-RNA complexes and their quantitative analysis via mass spectrometry. In this proposal, we apply this new technology to investigate how RNA-targeting drugs such as risdiplam change protein-RNA interactions and how this might relate to their mode-of-action in vivo.These research questions will be approached by a three-tier work programme in which UV-LED crosslinking and XRNAX are used to I) characterize RNA-targeting compounds in the cell culture, II) derive the murine RNA-binding proteome, and III) quantify the effect of splicing modulators on protein-RNA interactions in mice. In the first work package we will quantify the dose and time-dependent effect of risdiplam and nine other RNA-targeting drugs in cultured human cells. This will highlight previously unrelated protein-RNA interactions, which could be important for the function of the drugs. In the second work package we will derive a catalogue of RNA-binding proteins from five mouse organs and quantify tissue-specific differences. Finally, in the third work package we will elucidate the in vivo effect of risdiplam on protein-RNA interactions and compare it to another SMN2 splicing modulator called branaplam. We anticipate that a dose-dependent comparison between the two compounds might reveal commonly affected protein-RNA interactions, which in turn might explain their related specificity. The data will be made available for exploration online on ProteomicsDB.In summary, in the proposed project we apply UV-crosslinking proteomics for the study of RNA-targeting drugs in the cell culture and the mouse model. We believe that this will be of great benefit to our general understanding of protein-RNA interactions and the scientific community studying them.

• DFG Programme: Research Grants