Terminal uridylyltransferase 4
RNA uridylyltransferase activity
Kinetic and Mechanistic Studies of the Terminal Uridylyltransferase, Zcchc11 (TUT4).
Zcchc11 (TUT4, TENT3A, Z11) is a nucleotidyltransferase that catalyzes the 3'-polyuridylation of RNA. Our interest in this enzyme stems from its role in blocking the biogenesis of <i>let</i>-7, a family of microRNAs whose members act as tumor suppressors. Z11 polyuridylates pre-<i>let</i>-7, the precursor of <i>let</i>-7, when pre-<i>let</i>-7 is complexed with LIN28, an RNA-binding protein. Polyuridylation of pre-<i>let</i>-7 marks it for degradation. In addition to this LIN28-dependent activity, Z11 also has LIN28-independent activities. In this paper, we report the results of experiments that characterize LIN28-independent activities of Z11. Significant observations include the following. (1) Z11 uridylates not only mature <i>let</i>-7 species but also substrates as small as dinucleotides. (2) For both <i>let</i>-7i and the diribonucleotide AG, Z11 follows a steady-state ordered mechanism, with UTP adding before RNA. (3) Uridylation kinetics of <i>let</i>-7i (UGAGGUAGUAGUUUGUGCUGUU) and two truncated derivatives, GCUGUU and UU, indicate that Z11 manifests selectivity in <i>K</i><sub>m,RNA</sub>; <i>k</i><sub>cat,RNA</sub> values for the three substrates are nearly identical. (4) Z11 preferentially uridylates RNA lacking base-pairing near the 3' terminus. (5) Selectivity of Z11 toward ribonucleoside triphosphates is similar for <i>let</i>-7i and AG, with XTP preference: UTP > CTP > ATP ≫ GTP. Selectivity is manifested in <i>K</i><sub>m,XTP</sub>, with <i>k</i><sub>cat,XTP</sub> values being similar for UTP, CTP, and ATP. (6) Kinetic parameters for RNA turnover are dependent on the structure of the nucleoside triphosphate, consistent with recent structural data indicating stacking of the nucleoside triphosphate base with the base of the 3'-nucleotide of the substrate RNA (Faehnle et al., <i>Nat. Struct. Mol. Biol.</i> <b>2017</b>, <i>24</i>, 658).