A common tRNA modification at an unusual location: the discovery of wyosine biosynthesis in mitochondria.

Paul J Sample,Julius Lukeš,Luděk Kořený,Zdeněk Paris,Scott Hinger,Juan D Alfonzo,Eva Horáková,Kirk W Gaston,Ian M.C Fleming,Patrick A Limbach,Mary Anne T Rubio

doi:10.1093/nar/gkv286

Abstract

Establishment of the early genetic code likely required strategies to ensure translational accuracy and inevitably involved tRNA post-transcriptional modifications. One such modification, wybutosine/wyosine is crucial for translational fidelity in Archaea and Eukarya; yet it does not occur in Bacteria and has never been described in mitochondria. Here, we present genetic, molecular and mass spectromery data demonstrating the first example of wyosine in mitochondria, a situation thus far unique to kinetoplastids. We also show that these modifications are important for mitochondrial function, underscoring their biological significance. This work focuses on TyW1, the enzyme required for the most critical step of wyosine biosynthesis. Based on molecular phylogeny, we suggest that the kinetoplastids pathways evolved via gene duplication and acquisition of an FMN-binding domain now prevalent in TyW1 of most eukaryotes. These findings are discussed in the context of the extensive U-insertion RNA editing in trypanosome mitochondria, which may have provided selective pressure for maintenance of mitochondrial wyosine in this lineage.

Highlights

A defining feature of all tRNAs is the presence of numerous post-transcriptional chemical modifications [1,2]
Because some modifications are common to all tRNAs in all domains of life, it has been suggested that such ‘primordial’ nucleosides were essential in ensuring reading-frame maintenance early in the evolution of translational systems [3,4,5,6,7]
To further address the possibility of a wyosine biosynthetic pathway in mitochondria, we concentrated on the down regulation of TYW1L and TYW1S expression. We focused on these genes with the expectation that down-regulation of TYW1L would lead to disappearance of OHyW in the cytoplasm and mitochondrial fractions, in agreement with its synthesis in the cytosol followed by import of the hydroxywybutosine-containing tRNAs into the organelle; whereas down-regulation of TYWS would only affect formation of imG

Summary

Introduction

A defining feature of all tRNAs is the presence of numerous post-transcriptional chemical modifications [1,2]. Many positions in a tRNA can affect translational accuracy, position 37 of the anticodon loop plays an important, if not a critical, role in readingframe maintenance [6,8,9]. This universally modified position may harbor simple base methylations (such as 1methylguanosine, m1G), which in the context of a specific tRNA anticodon and depending on the inherent stability of anticodon-codon interactions, may be sufficient to prevent translational errors. One of the most chemically intricate modifications that occur at position 37 of the anticodon loop involves the nucleoside wyosine (imG) and its derivatives, including wybutosine (yW) and hydroxywybutosine (OHyW) [5]. Formation of m1G37 is followed by the incorporation of the C-1 and C-2 carbons of pyruvate by the

Methods

Results

Conclusion