NAD+-dependent synthesis of a 5-phospho-ADP-ribosylated RNA/DNA cap by RNA 2-phosphotransferase Tpt1

Abstract

RNA 2-phosphotransferase Tpt1 converts an internal RNA 2-monophosphate to a 2-OH via a two-step NAD+-dependent mechanism in which: (i) the 2-phosphate attacks the C1 of NAD+ to expel nicotinamide and form a 2-phospho-ADP-ribosylated RNA intermediate; and (ii) the ADP-ribose O2 attacks the phosphate of the RNA 2-phospho-ADPR intermediate to expel the RNA 2-OH and generate ADP-ribose 1–2 cyclic phosphate. Tpt1 is an essential component of the fungal tRNA splicing pathway that generates a unique 2-PO4, 3-5 phosphodiester splice junction during tRNA ligation. The wide distribution of Tpt1 enzymes in taxa that have no fungal-type RNA ligase raises the prospect that Tpt1 might catalyze reactions other than RNA 2-phosphate removal. A survey of Tpt1 enzymes from diverse sources reveals that whereas all of the Tpt1 enzymes are capable of NAD+-dependent conversion of an internal RNA 2-PO4 to a 2-OH (the canonical Tpt1 reaction), a subset of Tpt1 enzymes also catalyzed NAD+-dependent ADP-ribosylation of an RNA or DNA 5-monophosphate terminus. Aeropyrum pernix Tpt1 (ApeTpt1) is particularly adept in this respect. One-step synthesis of a 5-phospho-ADP-ribosylated cap structure by ApeTpt1 (with no subsequent 5-phosphotransferase step) extends the repertoire of the Tpt1 enzyme family and the cata-logue of ADP-ribosylation reactions involving nucleic acid acceptors.