Project Details
Projekt
Structure and function of RNA cytidine protonation
Subject Area
Biochemistry
Term
since 2026
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 583887882
Cytidine protonation (rC⁺) represents a reversible non-covalent RNA modification that can remodel base pairing and catalysis, yet its prevalence and biological functions in mammalian cells are largely unknown. By pyridine–borane (PyBo) sequencing, we recently discovered thousands of reactive cytidines in mouse embryonic stem cell RNA, strongly enriched in a consensus motif located in RNA sequence predicted to adopt hairpin loops. Concordantly, by NMR analysis we validated that consensus motif cytidine has a strongly shifted pKₐ of ~6.4. Altogether, the results indicate that PyBo-reactive cytidine residues can be protonated near physiological pH, potentially behaving as conditional epitranscriptomic marks. This proposal aims to elucidate the structural principles, chemical detectability, and biological readout of cytidine protonation. In Aim 1, the Schwalbe lab will determine high-resolution NMR structures of a candidate rC⁺ RNA its protonated and unprotonated states and compare them to mutants, revealing how rC⁺ reshapes local RNA architecture. In Aim 2, the Niehrs lab will test whether PyBo universally reacts with rC⁺, using model RNAs and riboswitches at controlled pH to define the chemical specificity and limitations of PyBo-based detection. In Aim 3, we will identify RNA-binding proteins that recognize rC⁺ elements. Finally, Aim 4 will assess the functional impact of the Inppl1 rC⁺ site on translation and mRNA stability using a dual-fluorescence ribosome stalling reporter in mammalian cells.
DFG Programme
Research Grants
