Editing and methylation at a single site by functionally interdependent activities

• Published in: Nature (London) Vol. 542; no. 7642; pp. 494 - 497
• Main Authors: Rubio, Mary Anne T., Gaston, Kirk W., McKenney, Katherine M., Fleming, Ian M. C., Paris, Zdeněk, Limbach, Patrick A., Alfonzo, Juan D.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23.02.2017; Nature Publishing Group
• Subjects: 631/326/417/1716; 631/337/1645/2570; 631/45/2783; 631/45/500; Anticodon - metabolism; Base Sequence; Biochemistry; Biosynthesis; Cytosine - analogs & derivatives; Cytosine - metabolism; Deamination; Enzymatic activity; Enzymes; Humanities and Social Sciences; letter; Methylation; Methyltransferases - metabolism; multidisciplinary; Mutagenesis; Mutagenicity; Nucleic acids; Nucleoside Deaminases - metabolism; Observations; Physiological aspects; Ribonucleic acid; RNA; RNA Editing; RNA, Transfer, Thr - chemistry; RNA, Transfer, Thr - genetics; RNA, Transfer, Thr - metabolism; Science; Trypanosoma brucei brucei - enzymology; Trypanosoma brucei brucei - genetics; Uridine - metabolism
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature21396

The C-to-U deamination at position 32 of tRNA Thr in Trypanosoma brucei requires two enzymatic activities and proceeds via formation of a 3-methylcytosine intermediate, supporting the notion of a coupled modification system. RNA methylation doubles up The array of modifications that can occur in DNA and RNA is much larger than is commonly appreciated, with more than 100 known modifications. However, few of these are easily replicated in vitro , leading to a proposal that coupling of modifications might be required. Juan Alfonzo and colleagues describe such a coupled system in the parasite Trypanosoma brucei . They find that the cytosine-to-uridine deamination at position 32 of tRNA Thr requires two enzymatic activities, and proceeds via the formation of a 3-methylcytosine (m 3 C) intermediate. In addition, the presence of the m 3 C methyltransferase, TRM140a, limits the potentially mutagenic activity of the deaminase ADAT2/3. Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified 1 . Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro . Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways 2 . A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro 3 . This model was prompted by the earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes, a reaction that has not been recapitulated in vitro and the mechanism of which remains unknown. Here we show that cytosine 32 in the anticodon loop of Trypanosoma brucei tRNA Thr is methylated to 3-methylcytosine (m 3 C) as a pre-requisite for C-to-U deamination. Formation of m 3 C in vitro requires the presence of both the T. brucei m 3 C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m 3 C is deaminated to 3-methyluridine (m 3 U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme 4 , but we also show that when co-expressed with the methyltransferase its mutagenicity is kept in check. This helps to explain how T. brucei escapes ‘wholesale deamination’ 5 of its genome while harbouring both enzymes in the nucleus. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.