RNA editing is a process that results in the production of a messenger RNA with nucleotide sequences that differ from those of the template DNA, and provides another mechanism for modulating gene expression. The phenomenon was initially described in the mitochondria of protozoa. Here we report that RNA editing is also required for the correct expression of plant mitochondrial genes. It has previously been proposed that in plant mitochondria there is a departure from the universal genetic code, with CGG specifying tryptophan instead of arginine. This was because CGG codons are often found in plant mitochondrial genes at positions corresponding to those encoding conserved tryptophans in other organisms. We have now found, however, wheat mitochondrial gene sequences containing C residues that are edited to U residues in the corresponding mRNA sequences. In this way, CGG codons can be changed to UGG codons in the mRNA so that tryptophan may be encoded according to the universal genetic code. Furthermore, for each codon modification resulting from a C----U conversion that we studied, we found a corresponding change in the amino acid that was encoded. RNA editing in wheat mitochondria can thus maintain genetic information at the RNA level and as a result contribute to the conservation of mitochondrial protein sequences among plants.

Very closely related short sequences are present at the 5′ end of cytoplasmic mRNAs in Euglena as evidenced by comparison of cDNA sequences and hybrid‐arrested translation experiments. By cloning Euglena gracilis nuclear DNA and isolating the rbcS gene (encoding the small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase), we have shown that the short leader sequence does not flank the nuclear gene sequence. The leader sequences were found to constitute the 5′ extremities of a family of small RNAs. Sequencing six members of this family revealed a striking similarity to vertebrate U snRNAs. We propose that a trans‐splicing mechanism transfers the spliced leader (SL) sequence from these small RNAs (SL RNAs) to pre‐mature mRNAs. Transfer of leader sequences to mRNAs by trans‐splicing has been shown only in trypanosomes where cis‐splicing is unknown, and in nematodes where not more than 10% of the mRNAs have leader sequences. Our results strongly suggest that Euglena is a unique organism in which both a widespread trans‐splicing and a cis‐splicing mechanism co‐exist.

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The small subunit (SSU) of ribulose 1‐5 bisphosphate carboxylase/oxygenase is a 15 kd protein in Euglena gracilis. The protein is synthesized as a 130 kd precursor as shown by immunoprecipitation of in vitro translation products and confirmed by immunoprecipitation of in vivo pulse‐labeled Euglena proteins. From the published SSU amino acid sequence, an oligonucleotide was synthesized that specifically hybridizes to a large mRNA whose length (approximately 4.3 kb) is consistent with the precursor size. The complete nucleotide sequence of the SSU mRNA was obtained by sequencing a cDNA clone from a lambda gt11 library and completed by direct mRNA sequencing. We report for the first time the complete sequence of a large mRNA and show that it encodes eight consecutive SSU mature molecules. The deduced precursor amino acid sequence shows that the amino terminus of the first SSU molecule is preceded by a 134 amino acid peptide which is cleaved during the maturation process. This long transit peptide exhibits features characteristic of signal peptides involved in the secretion of proteins through the endoplasmic reticulum. This is in agreement with the idea that the third (outer) membrane of the Euglena chloroplast envelope is of endoplasmic reticulum origin.

Evidence that nuclear‐encoded RNAs are present inside mitochondria has been reported from a wide variety of organisms, and is presumed to be due to import of specific cytosolic RNAs. In plants, the first examples were the mitochondrial leucine transfer RNAs of bean. In all cases, the evidence is circumstantial, based on hybridization of the mitochondrial RNAs to nuclear and not mitochondrial DNA. Here we show that transgenic potato plants carrying a leucine tRNA gene from bean nuclear DNA contain RNA transcribed from the introduced gene both in the cytosol and inside mitochondria, providing proof that the mitochondrial leucine tRNA is derived from a nuclear gene and imported into the mitochondria. The same bean gene carrying a 4 bp insertion in the anticodon loop was also expressed in transgenic potato plants and the transcript found to be present inside mitochondria, suggesting that this natural RNA import system could eventually be used to introduce foreign RNA sequences into mitochondria.

The complete cDNA sequence corresponding to the wheat coxIII gene transcript (coding for subunit 3 of cytochrome oxidase) has been determined by a method involving cDNA synthesis using specific oligonucleotides as primers followed by PCR amplification, cloning and sequencing of the amplification products. In 12 different clones, the same 13 nucleotide modifications have been found as compared to the genomic mitochondrial DNA sequence. Among these modifications, 12 are C----U conversions which change codons identities, thereby increasing the homology between the wheat COXIII protein and the corresponding protein of non-plant organisms. The 13th modification is a silent U----C conversion which seems to be an unfrequent editing eventin plant mitochondria. Homologies can be found between sequences surrounding editing sites in the coxIII transcript and in other wheat mitochondrial transcripts. The presence of such homology suggests that these sequences could base-pair with a common RNA molecule which might be involved in editing site recognition.

Four P. Vulgaris mitochondrial tRNA(Leu) species have been shown to be nuclear encoded. These mt tRNAs(Leu) can be used for in vitro protein synthesis. We found that the sequences of P. vulgaris mitochondrial and cytoplasmic tRNAs(Leu)(NAG) are identical except for a post-transcriptional modification occurring at position 18 (Gm in mt tRNA(Leu) instead of G in cyt tRNA(Leu], as in the case of mt and cyt tRNAs(Leu)(NAA) already sequenced. This post-transcriptional modification has also been found in two other bean mt tRNA(Leu) species, but not in P. vulgaris cytoplasmic tRNA(Leu) species that we have purified so far. Furthermore, comparison of the 2-D polyacrylamide gel electrophoretic patterns of tRNAs eluted from bean mt tRNA-mtDNA and mt tRNA-nDNA hybrids revealed at least 8 mt tRNAs coded for by the nuclear genome.

Total transfer RNAs were extracted from highly purified potato mitochondria. From quantitative measurements, the in vivo tRNA concentration in mitochondria was estimated to be in the range of 60 microM. Total potato mitochondrial tRNAs were fractionated by two-dimensional polyacrylamide gel electrophoresis. Thirty one individual tRNAs, which could read all sense codons, were identified by aminoacylation, sequencing or hybridization to specific oligonucleotides. The tRNA population that we have characterized comprises 15 typically mitochondrial, 5 'chloroplast-like' and 11 nuclear-encoded species. One tRNA(Ala), 2 tRNAs(Arg), 1 tRNA(Ile), 5 tRNAs(Leu) and 2 tRNAs(Thr) were shown to be coded for by nuclear DNA. A second, mitochondrial-encoded, tRNA(Ile) was also found. Five 'chloroplast-like' tRNAs, tRNA(Trp), tRNA(Asn), tRNA(His), tRNA(Ser)(GGA) and tRNA(Met)m, presumably transcribed from promiscuous chloroplast DNA sequences inserted in the mitochondrial genome, were identified, but, in contrast to wheat (1), potato mitochondria do not seem to contain 'chloroplast-like' tRNA(Cys) and tRNA(Phe). The two identified tRNAs(Val), as well as the tRNA(Gly), were found to be coded for by the mitochondrial genome, which again contrasts with the situation in wheat, where the mitochondrial genome apparently contains no tRNA(Val) or tRNA(Gly) gene (2).