Nucleotide specificity of the human terminal nucleotidyltransferase Gld2 (TUT2).
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.
Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
Christina Z
CZ
Chung
Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
David Hyung Suk
DH
Jo
Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
Ilka U
IU
Heinemann
D005075:Biological Evolution; D057809:HEK293 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism*; D011062:Polynucleotide Adenylyltransferase; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics / Q000378:metabolism*
D009711:Nucleotides; D011994:Recombinant Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D011062:Polynucleotide Adenylyltransferase; C550174:TENT2 protein, human
adenylation; miRNA; miRNA maturation; nucleotidyltransferase; uridylation