Two forms of RNA editing are required for tRNA maturation in Physarum mitochondria

Abstract

The mitochondrial genome of Physarum polycephalum encodes five tRNAs, four of which are edited by nucleotide insertion. Two of these tRNAs, tRNA(met1) and tRNA(met2), contain predicted mismatches at the beginning (proximal end) of the acceptor stem. In addition, the putative 5' end of tRNA(met2) overlaps the 3' end of a small, abundant, noncoding RNA, which we term ppoRNA. These anomalies led us to hypothesize that these two Physarum mitochondrial tRNAs undergo additional editing events. Here, we show that tRNA(met1) and tRNA(met2) each has a nonencoded G at its 5' end. In contrast to the other nucleotides that are added to Physarum mitochondrial RNAs, these extra G residues are likely added post-transcriptionally based on (1) the absence of added G in precursor transcripts containing inserted C and AA residues, (2) the presence of potential intermediates characteristic of 5' replacement editing, and (3) preferential incorporation of GTP into tRNA molecules under conditions that do not support transcription. This is the first report of both post-transcriptional nucleotide insertions and the addition of single Gs in P. polycephalum mitochondrial transcripts. We postulate that tRNA(met1) and tRNA(met2) are acted upon by an activity similar to that present in the mitochondria of certain other amoebozoons and chytrid fungi, suggesting that enzymes that repair the 5' end of tRNAs may be widespread.

FIGURE 1.

5′ RNA editing is predicted for two of the five tRNAs encoded in the mitochondrial genome of Physarum polycephalum. (A) Mitochondrial tRNA^met1 and tRNA^met2 each contain a mismatch at the beginning of the acceptor stem (Antes et al. 1998). Sites predicted to contain a G residue in the mature tRNA products are indicated with arrows; sites of internal C insertions identified by Antes et al. (1998) are indicated with arrowheads. (B) Apparent overlap of the 5′ end of tRNA^met2 with the 3′ end of ppoRNA. The approximate positions of primers used for reverse transcription (gray) and PCR (black) of nascent transcripts are shown.

FIGURE 2.

Editing at the 5′ ends of tRNA^met1 and tRNA^met2. Sequence of representative cloned RT-PCR products derived from circularized tRNAs; traces of the region between the primers used for PCR amplification are shown. The nonencoded CCA ends (italics) are underlined, and the 5′ ends of the mature tRNAs are indicated by green arrows in the traces. Internal C insertion sites are indicated with blue asterisks. (A) The acceptor stem of tRNA^met1 and traces from clones with a G at its 5′ end (edited, top) or missing the 5′ nucleotide (potential editing intermediate, bottom). Both contain the extra C that is added in this region. (B) Acceptor stem of tRNA^met2 and traces from tRNA^met2 clones having a 5′ G (edited, top) or C (unedited, bottom). (C) Clone from circularized tRNA^pro containing the encoded C at its 5′ end and internal C insertions.

FIGURE 3.

No extra G is present at the ppoRNA-tRNA^met2 boundary in RNA precursors containing added nucleotides at other positions. Sequence traces of bulk PCR and RT-PCR products derived from mitochondrial DNA (A) and nascent transcripts (B) containing the 3′ end of the mitochondrial small subunit rRNA (SSU), ppoRNA, tRNA^met2, and tRNA^lys. The region encoding the 3′ portion of ppoRNA (blue line) and the 5′ portion of tRNA^met2 (red line) is enlarged here. The traces from which these are extracted are shown in Supplemental Figure S1. The site of potential overlap of the ppoRNA and tRNA^met2 is indicated by a black arrow, and the nonencoded C inserted in this portion of the ppoRNA is marked with a blue asterisk.

FIGURE 4.

Processed tRNAs are preferentially labeled with [α³²P]GTP in isolated mitochondria. Isolated mitochondria were incubated in transcription buffer (lanes 2,3), the optimal buffer for the A. castellanii 5′ editing activity (lanes 4,5), or a modified version of the A. castellanii buffer (lanes 6,7), in the presence of [α-³²P]GTP (lanes 2,4,6) or [α-³²P]UTP (lanes 3,5,7) as described in Materials and Methods. Lane 8 shows the position of mature tRNAs labeled with [α-³²P]ATP by the CCA-adding activity present in S100 extracts derived from Physarum mitochondria. End-labeled, denatured DNA size markers are shown in lane 1.