Biochemical and structural bioinformatics studies of fungal CutA nucleotidyltransferases explain their unusual specificity towards CTP and increased tendency for cytidine incorporation at the 3′-terminal positions of synthesized tails.

Non-canonical RNA nucleotidyltransferases (NTases), including poly(A), poly(U) polymerases (PAPs/PUPs) and C/U-adding enzymes, modify 3′-ends of different transcripts affecting their functionality and stability. They contain PAP/OAS1 substrate-binding domain (SBD) with inserted NTase domain. Aspergillus nidulans CutA (AnCutA), synthesizes C/U-rich 3′-terminal extensions in vivo Here, using high-throughput sequencing of the 3′-RACE products for tails generated by CutA proteins in vitro in the presence of all four NTPs, we show that even upon physiological ATP excess synthesized tails indeed contain an unprecedented number of cytidines interrupted by uridines and stretches of adenosines, and that majority end with two cytidines. Strikingly, processivity assays documented that in the presence of CTP as a sole nucleotide, the enzyme terminates after adding two cytidines only. Comparison of our CutA 3D model to selected non-canonical NTases of known structures revealed substantial differences in the nucleotide recognition motif (NRM) within PAP/OAS1 SBD. We demonstrate that CutA specificity towards CTP can be partially changed to PAP or PUP by rational mutagenesis within NRM and, analogously, Cid1 PUP can be converted into C/U-adding enzyme. Collectively, we suggest that a short cluster of amino acids within NRM is a determinant of NTases’ substrate preference, which may allow to predict their specificity.