Unusual Noncanonical Intron Editing Is Important for tRNA Splicing in Trypanosoma brucei

Molecular Cell

Available online 3 October 2013

Mary Anne T. Rubio, Zdeněk Paris, Kirk W. Gaston, Ian M.C. Fleming, Paul Sample, Christopher R. Trotta, Juan D. Alfonzo.

¹ Department of Microbiology and The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
² Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA
³ PTC Therapeutics, 100 Corporate Court, South Plainfield, NJ 07080, USA

Design acknowledgments: Mary Anne Rubio, Juan Alfonzo, Chris Trotta, & Craig Foster

In cells, tRNAs are synthesized as precursor molecules bearing extra sequences at their 5′ and 3′ ends. Some tRNAs also contain introns, which, in archaea and eukaryotes, are cleaved by an evolutionarily conserved endonuclease complex that generates fully functional mature tRNAs. In addition, tRNAs undergo numerous posttranscriptional nucleotide chemical modifications. In Trypanosoma brucei, the single intron-containing tRNA (tRNA^Tyr_GUA) is responsible for decoding all tyrosine codons; therefore, intron removal is essential for viability. Using molecular and biochemical approaches, we show the presence of several noncanonical editing events, within the intron of pre-tRNA^Tyr_GUA, involving guanosine-to-adenosine transitions (G to A) and an adenosine-to-uridine transversion (A to U). The RNA editing described here is required for proper processing of the intron, establishing the functional significance of noncanonical editing with implications for tRNA processing in the deeply divergent kinetoplastid lineage and eukaryotes in general.

The T. brucei TRM5 methyltransferase plays an essential role in mitochondrial protein synthesis and function.

 

 

 

RNA. 2013 Mar 21. 

Paris Z, Horáková E, Rubio MA, Sample P, Fleming IM, Armocida S, Lukes J, Alfonzo JD.

Department of Microbiology, Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.

Abstract

All tRNAs undergo post-transcriptional chemical modifications as part of their natural maturation pathway. Some modifications, especially those in the anticodon loop, play important functions in translational efficiency and fidelity. Among these, 1-methylguanosine, at position 37 (m¹G₃₇) of the anticodon loop in several tRNAs, is evolutionarily conserved and participates in translational reading frame maintenance. In eukaryotes, the tRNA methyltransferase TRM5 is responsible for m¹G formation in nucleus-encoded as well as mitochondria-encoded tRNAs, reflecting the universal importance of this modification for protein synthesis. However, it is not clear what role, if any, mitochondrial TRM5 serves in organisms that do not encode tRNAs in their mitochondrial genomes. These organisms may easily satisfy the m¹G₃₇ requirement through their robust mitochondrial tRNA import mechanisms. We have explored this possibility in the parasitic protist Trypanosoma brucei and show that down-regulation of TRM5 by RNAi leads to the expected disappearance of m¹G₃₇, but with surprisingly little effect on cytoplasmic translation. On the contrary, lack of TRM5 causes a marked growth phenotype and a significant decrease in mitochondrial functions, including protein synthesis. These results suggest mitochondrial TRM5 may be needed to mature unmethylated tRNAs that reach the mitochondria and that could pose a problem for translational fidelity. This study also reveals an unexpected lack of import specificity between some fully matured and potentially defective tRNA species.