Nucleotide specificity of the human terminal nucleotidyltransferase Gld2 (TUT2)

Abstract

The nontemplated addition of single or multiple nucleotides to RNA transcripts is an efficient means to control RNA stability and processing. Cytoplasmic RNA adenylation and the less well-known uridylation are post-transcriptional mechanisms regulating RNA maturation, activity, and degradation. Gld2 is a member of the noncanonical poly(A) polymerases, which include enzymes with varying nucleotide specificity, ranging from strictly ATP to ambiguous to exclusive UTP adding enzymes. Human Gld2 has been associated with transcript stabilizing miRNA monoadenylation and cytoplasmic mRNA polyadenylation. Most recent data have uncovered an unexpected miRNA uridylation activity, which promotes miRNA maturation. These conflicting data raise the question of Gld2 nucleotide specificity. Here, we biochemically characterized human Gld2 and demonstrated that it is a bona fide adenylyltransferase with only weak activity toward other nucleotides. Despite its sequence similarity with uridylyltransferases (TUT4, TUT7), Gld2 displays an 83-fold preference of ATP over UTP. Gld2 is a promiscuous enzyme, with activity toward miRNA, pre-miRNA, and polyadenylated RNA substrates. Apo-Gld2 activity is restricted to adding single nucleotides and processivity likely relies on additional RNA-binding proteins. A phylogeny of the PAP/TUTase superfamily suggests that uridylyltransferases, which are derived from distinct adenylyltransferase ancestors, arose multiple times during evolution via insertion of an active site histidine. A corresponding histidine insertion into the Gld2 active site alters substrate specificity from ATP to UTP.

Biochemical and structural bioinformatics studies of fungal CutA nucleotidyltransferases explain their unusual specificity toward CTP and increased tendency for cytidine incorporation at the 3′-terminal positions of synthesized tails

“…On the contrary, S. cerevisiae Trf4 and Trf5 proteins, displaying PAP activity (LaCava et al 2005;Vanacova et al 2005;Wyers et al 2005;Houseley and Tollervey 2006;Hamill et al 2010), contain DxxDxxN cluster at the equivalent position and the same is true for S. pombe nuclear noncanonical PAP, Cid14 (Table 1; Win et al 2006). Importantly, asparagine seems to be invariantly present in the last position of the NRM cluster in all noncanonical NTases, for which PAP activity has been experimentally demonstrated, including recently studied human GLD-2 (TUT2) which contains ExxDxxN cluster (Table 1; Chung et al 2016). In concordance, the Cid1 H336N mutation (creating DxxExxN cluster) changed Cid1 specificity from PUP into PAP (Lunde et al 2012).…”

Unbiased screen of RNA tailing enzymes at single-nucleotide resolution reveals a poly(UG) polymerase required for genome integrity and RNA silencing

“…The active site regions of the PAPs and PUPs we identified bear on how U and A are distinguished by different enzymes in the same family. Prior work demonstrated that a histidine in the active-site regions of Human Gld2 and S. pombe Cid1 dictates their apparent preferences for A and U, respectively [62][63][64][65][66][67] . Similarly, U-adding enzymes appear to have arisen repeatedly in evolution by the insertion of histidine into ancestral A-adding enzymes 62 .…”

“…Prior work demonstrated that a histidine in the active-site regions of Human Gld2 and S. pombe Cid1 dictates their apparent preferences for A and U, respectively [62][63][64][65][66][67] . Similarly, U-adding enzymes appear to have arisen repeatedly in evolution by the insertion of histidine into ancestral A-adding enzymes 62 . However, among the U-adding enzymes we uncovered here, several (Sp Cid16, Ce PUP-3 and Ce F43E2.1) lack that histidine, and one that possesses a histidine (Hs TUT1/Star-PAP) adds adenosines 21 (Table 1).…”

RNA uridylation: a key posttranscriptional modification shaping the coding and noncoding transcriptome

“…In addition, a histidine residue in Cid1 (His336), conserved in some plant and human cytosolic TUTases but absent from trypanosomal TUTases, has been involved in UTP selectivity . A single amino acid substitution at this position is sufficient to switch the specificity of Cid1 from UTP to ATP, and vice versa for human Gld2 (TUT2) . Those experiments illustrate the ease of switching the nucleotide specificity of a terminal nucleotidyltransferase during evolution to allow for the acquisition of a novel biological function linked to RNA tailing.…”