Editing and methylation at a single site by functionally interdependent activities

Abstract

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 modified1. 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 pathways2. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro3. 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 tRNAThr is methylated to 3-methylcytosine (m3C) as a pre-requisite for C-to-U deamination. Formation of m3C in vitro requires the presence of both the T. brucei m3C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m3C is deaminated to 3-methyluridine (m3U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme4, 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.

Dynamic RNA Modifications in Gene Expression Regulation

“…Complex modifications often require step-wise installation by a cascade of enzymes (e.g. wybutosine and mcm 5 s 2 U) or methylation followed by deamination of the same base, a form of RNA editing (Rubio et al, 2017). …”

To be or not to be modified: Miscellaneous aspects influencing nucleotide modifications in tRNAs

“…d) ; and the I34 modification introduced in Trypanosoma brucei on tRNA Thr(AGU) by ADAT2/ADAT3 heterodimer, which is stimulated by prior deamination of C32 leading to U32, which is itself stimulated by the prior methylation of C32 leading to m 3 C32 (Fig. e) . In all these cases, the precise molecular mechanisms that define these modification circuits are not yet fully understood, but it is reasonable to conceive that the prior modification might either act as a direct recognition determinant for the subsequent modification enzyme or alter the local structure of the anticodon‐loop region, thereby helping to present the appropriate structure to the subsequent modification enzyme.…”

Understanding RNA modifications: the promises and technological bottlenecks of the ‘epitranscriptome’

“…In addition, modifications, if not reversed to the unmodified state, can be further modified such as, for instance, Ψ by N1 methylation [39], m 5 C to various oxidation products by the activity of ten-eleven translocation family enzymes [40] or 3-methylcytosine (m 3 C) to 3-methyluridine (m 3 U) [41]. …”