Novel ribonuclease activity of cusativin from Cucumis sativus for mapping nucleoside modifications in RNA

A recombinant ribonuclease, cusativin, was characterized for its cytidine-specific cleavage ability of RNA to map chemical modifications. Following purification of native cusativin protein as described before (Rojo et al. Planta 194:328, 17), partial amino acid sequencing was carried out to identify the corresponding protein coding gene in cucumber genome. Cloning and heterologous expression of the identified gene in Escherichia coli resulted in successful production of active protein as a C-terminal His-tag fusion protein. The ribonuclease activity and cleavage specificity of the fusion protein were confirmed with a variety of tRNA isoacceptors and total tRNA. Characterization of cusativin digestion products by ion-pairing reverse-phase liquid chromatography coupled with mass spectrometry (IP-RP-LC-MS) analysis revealed cleavage of CpA, CpG, and CpU phosphodiester bonds at the 3'-terminus of cytidine under optimal digestion conditions. Ribose methylation or acetylation of cytosine inhibited RNA cleavage. The CpC phosphodiester bond was also resistant to cusativin-mediated RNA cleavage; a feature to our knowledge has not been reported for other nucleobase-specific ribonucleases. Here, we demonstrate the analytical utility of such a novel feature for obtaining high-sequence coverage and accurate mapping of modified residues in substrate RNAs.