Reproducible and efficient new method of RNA 3'-end labelling by CutA nucleotidyltransferase-mediated CC-tailing

Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5MODIFIER LETTER PRIME or 3MODIFIER LETTER PRIME terminus of the molecule can serve as probes in hybridization-based analyses of in vivo degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases. In some applications, the placement of the label is not a concern, while in other cases it is required that the radioactive mark is located at the 5MODIFIER LETTER PRIME- or 3MODIFIER LETTER PRIME-end of the molecule. An unsurpassed method for 5MODIFIER LETTER PRIME-end RNA labelling employs T4 polynucleotide kinase (PNK) and [gamma-P-32]ATP. In the case of 3MODIFIER LETTER PRIME-end labelling, several different possibilities exist. However, they require the use of costly radionucleotide analogues. Previously, we characterized an untypical nucleotidyltransferase named CutA, which preferentially incorporates cytidines at the 3MODIFIER LETTER PRIME-end of RNA substrates. Here, we demonstrate that this unusual feature can be used for the development of a novel, efficient, reproducible and economical method of RNA 3MODIFIER LETTER PRIME-end labelling by CutA-mediated cytidine tailing. The labelling efficiency is comparable to that achieved with the most common method applied to date, i.e. [5MODIFIER LETTER PRIME-P-32]pCp ligation to the RNA 3MODIFIER LETTER PRIME-terminus catalysed by T4 RNA ligase I. We show the utility of RNA substrates labelled using our new method in exemplary biochemical assays assessing directionality of two well-known eukaryotic exoribonucleases, namely Dis3 and Xrn1.