USB1 directly catalyzes 3′ terminal 2′, 3′ cyclic phosphate formation. (A) Degradation of oligo(U)₇ RNA by Exo T (i) or USB1 (ii, iii). C: oligo(U)₇ RNA incubated in buffer for 30 minutes. (B) Expected masses of potential oligo(U)₇ RNA degradation products containing 3′OH, 3′p or 3′ > p groups. Blue box: observed masses after incubation with Exo T; red boxes: observed masses after incubation with USB1. (C) MALDI-TOF mass spectrometric analysis of oligo(U)₇ RNA degradation products. m/z, mass-charge ratio. ∧ indicates artifacts. USB1 degradation products with 3′ end > p modifications are marked in red.