In Vitro Characterization of a tRNA Editing Activity in the Mitochondria of Spizellomyces punctatus, a Chytridiomycete Fungus

Charles E Bullerwell,Michael W Gray

doi:10.1074/jbc.m411273200

Abstract

In the chytridiomycete fungus, Spizellomyces punctatus, all eight of the mitochondrially encoded tRNAs are predicted to have one or more base pair mismatches at the first three positions of their aminoacyl acceptor stems. These tRNAs are edited post-transcriptionally by replacement of the 5'-nucleotide in each mismatched pair with a nucleotide that can form a standard Watson-Crick base pair with its counterpart in the 3'-half of the stem. The type of mitochondrial tRNA editing found in S. punctatus also occurs in Acanthamoeba castellanii, a distantly related amoeboid protist. Using an S. punctatus mitochondrial extract, we have developed an in vitro assay of tRNA editing in which nucleotides are incorporated into various tRNA substrates. Experiments employing synthetic transcripts revealed that the S. punctatus tRNA editing activity incorporates nucleotides on the 5'-side of substrate tRNAs, uses the 3'-sequence as a template for incorporation, and adds nucleotides in a 3'-to-5' direction. This activity can add nucleotides to a triphosphorylated 5'-end in the absence of ATP but requires ATP to add nucleotides to a monophosphorylated 5'-end; moreover, it functions independently of the state of tRNA 3' processing. These data parallel results obtained in a previous in vitro study of A. castellanii tRNA editing, suggesting that remarkably similar activities function in the mitochondria of these two organisms. The evolutionary origins of these activities are discussed.

Highlights

RNA editing encompasses particular forms of RNA processing that change the primary sequence of an RNA molecule from that predicted by the gene sequence
Development of an in Vitro Assay of Nucleotide Incorporation—Intact mitochondria were recovered from S. punctatus zoospores by lysis under relatively mild conditions in a French pressure cell followed by differential centrifugation
An in Vitro Assay of Mitochondrial transfer RNAs (tRNAs) Editing in S. punctatus—We have obtained a mitochondrial extract from S. punctatus, a chyridiomycete fungus, that supports specific and efficient incorporation of nucleotides into natural and in vitrotranscribed tRNAs

Summary

Introduction

RNA editing encompasses particular forms of RNA processing that change the primary sequence of an RNA molecule from that predicted by the gene sequence. An additional type of editing, and the first example of tRNA editing to be described, occurs in the mitochondria of the amoeboid protist Acanthamoeba castellanii [31] In this organism, 12 of 15 mtDNA1-encoded tRNAs are predicted to contain mismatches in one or more of the first three base pairs of the acceptor stem. In Spizellomyces punctatus, a chytridiomycete fungus, all eight of the mtDNA-encoded tRNAs are predicted to contain mismatches at one or more of the first three acceptor stem positions [34] These mismatches are repaired in vivo, as in A. castellanii, by replacement of the 5Ј-nucleotide in the pair to form Watson-Crick base pairs, as shown by direct sequencing of tRNA 5Ј-ends [34] and tRNA circularization/RT-PCR [35]. Editing in S. punctatus is highly similar in effect to that in A. castellanii, a quite unexpected result considering the lack of a specific evolutionary relationship between chytridiomycete fungi and this amoeboid protist

Methods

Results

Conclusion