Poster abstracts

Poster number 44 submitted by Aubree Zimmer

tRNA recognition by the T. brucei methyltransferase Trm140

Aubree A. Zimmer (Ohio State Biochemistry Program, Department of Microbiology, OSU Center for RNA Biology), Katherine M. McKenney (Ohio State Biochemistry Program, Department of Microbiology, OSU Center for RNA Biology), Mary Anne T. Rubio (Department of Microbiology, OSU Center for RNA Biology), Juan D. Alfonzo (Ohio State Biochemistry Program, Department of Microbiology, OSU Center for RNA Biology)

Abstract:
Transfer RNAs (tRNAs) are central to protein synthesis by converting the information found in protein-coding regions of the genome into a precise polypeptide chain as dictated by the codons in mRNA. However, before becoming functional in translation, tRNAs are subject of several processing steps such as intron removal, CCA addition, cleavage of 5′ leader and 3′ trailer sequences, along with extensive chemical modifications necessary for proper function. For instance, the conversion of A to I at the wobble base of the anticodon increases decoding capabilities, while pseudouridine at position 55 is evolutionarily conserved and plays a key role in tRNA stability. While many tRNA modification enzymes across a wide variety of species have been described, how these enzymes target a specific tRNA substrate among a pool of similar non-substrate molecules is still unclear. In Trypanosoma brucei, we have described a methylation at position 32 of all three tRNAThr, which requires a deaminase (ADAT 2/3) and a methyltransferase (Trm140) working together while showing intertwining functionality to convert an encoded C32 to m3U32—in what we describe as enzyme co-activation. Our laboratory aims to understand enzyme co-dependence by first understanding the biochemical interactions between the two enzymes and tRNA. In vitro binding studies using both enzymes and tRNA mini substrates shows Trm140 and ADAT 2/3 can only bind synergistically to a complete tRNA cloverleaf. We also show both enzymes can bind non-substrate tRNA’s with equal affinity to substrate, but only modify tRNAThr. The results presented here aim at elucidating how the Trm140 methylase recognizes and binds its cognate tRNA substrate, highlighting nuances in substrate specificity by tRNA processing enzymes.

References:
1. Rubio MAT et al. 2017. Editing and methylation at a single site by functionally interdependent activities. Nature 542: 494–497
2. McKenney KM, Rubio MAT, Alfonzo JD. 2018. Binding synergy as an essential step for tRNA editing and modification enzyme codependence in Trypanosoma brucei. RNA 24: 56-66.
5. McKenney KM, 2018. Investigating the basis of tRNA editing and modification enzyme coactivation in Trypanosoma brucei. PhD Dissertation. The Ohio State University, OH.

Keywords: tRNA editing, Methyltransferase, Trypanosoma