Tpt1 is an essential agent of fungal and plant tRNA splicing that removes an internal RNA 2′-phosphate generated by tRNA ligase. Tpt1 also removes the 2′-phosphouridine mark installed by Ark1 kinase in the V-loop of archaeal tRNAs. Tpt1 performs a two-step reaction in which the 2′-PO
4
attacks NAD
+
to form an RNA-2′-phospho-(ADP-ribose) intermediate, and transesterification of the ADP-ribose O2″ to the RNA 2′-phosphodiester yields 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Here, we present structures of archaeal Tpt1 enzymes, captured as product complexes with ADP-ribose-1″-PO
4
, ADP-ribose-2″-PO
4
, and 2′-OH RNA, and as substrate complexes with 2′,5′-ADP and NAD
+
, that illuminate 2′-PO
4
junction recognition and catalysis. We show that archaeal Tpt1 enzymes can use the 2′-PO
4
-containing metabolites NADP
+
and NADPH as substrates for 2′-PO
4
transfer to NAD
+
. A role in 2′-phospho-NADP(H) dynamics provides a rationale for the prevalence of Tpt1 in taxa that lack a capacity for internal RNA 2′-phosphorylation.