C to U Editing Stimulates A to I Editing in the Anticodon Loop of a Cytoplasmic Threonyl tRNA in Trypanosoma brucei

Abstract

Editing of tRNAs is widespread in nature and either changes the decoding properties or restores the folding of a tRNA. Unlike the phylogenetically disperse adenosine (A) to inosine (I) editing, cytosine (C) to uridine (U) editing has only been previously described in organellar tRNAs. We have shown that cytoplasmic tRNA Thr (AGU) undergoes two distinct editing events in the anticodon loop: C to U and A to I. In vivo, every inosine-containing tRNA Thr is also C to U edited at position 32. In vitro, C to U editing stimulates conversion of A to I at the wobble base. Although the in vivo and in vitro requirements differ, in both cases, the C to U change plays a key role in A to I editing. Due to an unusual abundance of A34-containing tRNAs, our results also suggest that the unedited and edited tRNAs are functional, each dedicated to decoding a specific threonine codon. C to U editing of cytoplasmic tRNA expands the editing repertoire in eukaryotic cells, and when coupled to A to I changes, leads to an interrelation between editing sites.

Induced Resistance to Methionyl-tRNA Synthetase Inhibitors in Trypanosoma brucei Is Due to Overexpression of the Target

“…We assume that these changes translate into higher levels of expressed MetRS protein, although this was not directly measured. Two attempts were made to quantify the MetRS enzymatic activity of cell lysates from large cultures of T. brucei using a radiometric aminoacylation assay (20), but the method was not sufficiently sensitive to detect activity in any of the samples. We felt that the genetic evidence for gene amplification by Northern and Southern analyses was sufficiently compelling that the cost of preparing antibodies for Western blots was not justified.…”

tRNA biology charges to the front

“…Since mutation of the active site of ADAT2 eliminates both A-to-I editing of tRNA and C-to-U editing of DNA, while not preventing complex formation, it seems highly likely that the single active site contains both activities. Furthermore, at least in vivo, this same protein appears to be responsible for both the C-to-U editing that occurs at residue 32 of tRNA Thr and the A-to-I editing that occurs at residue 34 (Rubio et al 2006). These findings point to a common evolutionary origin of both classes of these editing activities, and suggest the possibility that both editing activities in tRNA processing derive from one activity (Rubio et al 2007).…”

The C-terminal end of the Trypanosoma brucei editing deaminase plays a critical role in tRNA binding

“…Labeled tRNA substrate was prepared as previously described by us (Rubio et al 2006). Radio-labeled substrate was first heated to 70°C for 3 min to denature the RNA and allowed to refold at room temp for 5 min in reaction buffer (50 mM Tris-HCl pH 8.0, 2.5 mM MgSO 4 , 0.1mM EDTA, and 1 mM DTT).…”

“…To date, an ever-growing number of proven and putative targets of deamination editing have been identified, including various tRNAs, mRNAs, and 7SL RNA (Alfonzo et al 1999;Ben-Shlomo et al 1999;Gott and Emeson 2000;Petersen-Mahrt et al 2002;Dickerson et al 2003;Rubio 2005;Rubio et al 2006). Deaminases are also responsible for free nucleotide interconversions that contribute to nucleotide pools in cells and serve as a repository for the nucleotide building blocks used in both RNA and DNA synthesis (Wolfenden 1993;Betts et al 1994).…”