Trypanosome Tpt1 is a bifunctional enzyme composed of tandem DEDDh nuclease and RNA 2'-phosphotransferase domains.

Abstract

RNA 2'-phosphotransferase Tpt1 is a widely distributed enzyme that removes an internal RNA 2'-phosphate by transfer to NAD+. Tpt1 is essential in fungi, where it erases the 2'-PO4 mark installed by tRNA ligase during tRNA splicing. Tpt1 enzymes from bacteria, archaea, fungi, and metazoa are monofunctional units composed of RNA-binding and NAD+-binding lobes. The lone exception to date is fission yeast Tpt1, which consists of an N-terminal RNA 2'-phosphotransferase catalytic domain fused to a C-terminal domain homologous to budding yeast iron-sulfur cluster assembly factor Yae1. Here we report that the Tpt1 homologs of kinetoplastid protozoa are bifunctional enzymes composed of an N-terminal DEDDh-type nuclease domain linked to a C-terminal 2'-phosphotransferase domain. Characterization of Trypanosoma brucei Tpt1 (TbrTpt1) revealed that: (i) the C-terminal domain TbrTpt1-(298-540) is an autonomous module that catalyzes 2'-PO4 transfer to NAD+ via an RNA-2'-phospho-(ADP-ribose) intermediate; (ii) TbrTpt1-(299-540) is active in tRNA splicing in vivo when expressed in budding yeast tpt1∆ cells; and (iii) TbrTpt1 is a manganese-dependent 3'-exonuclease adept at resecting DNA and RNA single strands. TbrTpt1's RNA exonuclease is blocked by a 2' terminal phosphate. TbrTpt1 and TbrTpt1-(298-540) can remove a 2'-terminal phosphate via transfer to NAD+, albeit much less efficiently than removal of an internal 2'-phosphate.