Poster abstracts
Poster number 12 submitted by Katie McKenney
An adenosine deaminase and methyltransferase act co-dependently to edit and modify tRNA in Trypanosoma brucei
Katherine M. McKenney (Department of Microbiology and OSU Center for RNA Biology), Ian M.C. Fleming (Department of Microbiology and OSU Center for RNA Biology), Kirk W. Gaston (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati), Pat A. Limbach (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati), Mary Anne Rubio (Department of Microbiology and OSU Center for RNA Biology), Juan D. Alfonzo (Department of Microbiology and OSU Center for RNA Biology)
Abstract:
In all domains of life, transfer RNAs (tRNAs) undergo a number of post-transcriptional modifications and editing that fine-tune their structural and decoding properties. These processing events can be indispensable for translation and consequently for survival. In T. brucei, tRNAThr undergoes hydrolytic deamination from adenosine (A) to inosine (I) at position 34. This deamination event expands the decoding capacity of tRNAThr by permitting wobble basepairing with the third position of the cognate codon. Along with the A to I editing, tRNAThr in T. brucei is edited from cytosine (C) to uridine (U) at position 32 of the anticodon loop. Both deamination events are uniquely catalyzed by the same enzyme, TbADAT2/3. Besides the deamination editing, the tRNAThr is further modified to 3-methylcytidine (m3C) at position 32. Our laboratory has identified two homologs of the Saccharomyces cerevisiae m3C methyltransferase in T. brucei, one of which has been validated to be the m3C methyltransferase (TbTrm140). Interestingly, in vitro assays have shown that recombinant TbTrm140 methylates C32 of a synthetic tRNA substrate only when TbADAT2/3 is in the reaction. Furthermore, deamination of m3U is observed in vitro only upon prior formation of m3C. These unprecedented results demonstrate that the methytransferase and deaminase depend on each other to carry out their respective functions. To this end we also show that the two enzymes act synergistically whereby the deaminase enhances the affinity of the methyltransferase for its substrate and vice versa. Additionally, co-immunoprecipitation data provide further evidence of complex formation. This interaction supports the idea that some modification enzymes act in a concerted manner to shape tRNA structure and function.
Keywords: Trypanosoma, tRNA editing, methylation