Fission yeast Tpt1 is composed of tandem RNA 2'-phosphotransferase and Yae1 domains, both of which are essential for viability.

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 executes a two-step reaction in which: (i) the RNA 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate and expel nicotinamide; and (ii) the ADP-ribose O2″ attacks the RNA 2'-phosphodiester to form 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. All Tpt1 enzymes studied to date are monofunctional units comprising a single bilobed fold composed of an RNA-binding lobe and an NAD+-binding lobe. We now find that fission yeast Tpt1 is an exception to this rule. Schizosaccharomyces pombe Tpt1 (SpTpt1) consists of an N-terminal RNA 2'-phosphotransferase catalytic domain (aa 1-237) linked to a C-terminal domain (aa 238-365) homologous to budding yeast iron-sulfur cluster assembly factor Yae1. The SpTpt1 catalytic domain and the Yae1 domain are both essential for S. pombe growth, though they need not be linked within the same polypeptide. A mutational analysis of the 2'-phosphotransferase domain illuminates the distinct contributions of essential active site constituents Arg50 and Arg96 during the two chemical steps of the Tpt1 pathway.