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

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 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.