Mol Gen Genet (1993) 240:445-449

© Springer-Verlag 1993

Short communications

Ribosomal protein gene rpl5 is eotranseribed with the had3 gene in Oenothera mitochondria Woifgang Schuster

Institut ffir Genbiologische Forschung GmbH, Ihnestrasse 63, D-14195 Berlin, Germany

Received: 29 March 1993/Accepted: 1 April 1993

Abstract. The rpl5 ribosomal protein gene was identified in the mitochondrial genome of the higher plant Oenothera berteriana. The gene is present in a unique genomic location upstream of the gene encoding subunit 3 of the NADH dehydrogenase (had3). Both genes are cotranscribed, and the mRNA is modified at several cy- tidine residues by RNA editing. Analysis of the editing profiles of both genes by direct cDNA analysis and poly- merase chain reaction (PCR) revealed that not all tran- scripts are fully edited at all sites. Eight of the nine C to U conversions in the rpl5 reading frame are non-silent and change the deduced amino acid sequence. The genes of the prokaryotic-like cistron that includes the rpsl9, rps3, rp116, rpl5, and rps14 genes, which is at least partial- ly conserved in the mitochondrial genomes of other higher and lower plants, are dispersed in the Oenothera mitochondrial genome.

Key words: RNA editing Plant mitochondria- Riboso- mal protein gene cluster

Introduction

Mitochondrial ribosomes are assembled from proteins encoded by different cellular compartments, while the ribosomal RNAs are encoded exclusively by the mito- chondrial genome. The eubacteria-like ancestors of mito- chondria brought their own translational system into the cell, but many of the genes encoding components of the translational apparatus have been transferred to the nu- cleus during evolution. Of the 60-80 assumed ribosomal protein genes, at most only a small portion has been retained in the mitochondrion. While vertebrates encode all ribosomal proteins in the nuclear compartment, the gene for one ribosomal protein has been found in the mitochondrial genome of yeast, but all other ribosomal proteins are imported from the cytoplasm as in animals

Communicated by R. Hagemann

(Butow et al. 1985). In the lower land plant Marchantia (liverwort), sixteen genes encoding ribosomal proteins have been found in the mitochondrial genome. In the chloroplast genomes of tobacco (Shinozaki et al. 1986), liverwort (Ohyama et al. 1986) and rice (Hiratsuka et al. 1989) about 20 genes encoding plastid ribosomal proteins have been located. The mtDNAs of higher plants also encode several ribosomal protein genes, some of which have been identified in the past few years: rps3 in maize (Hunt and Newton 1991), Oenothera (Schuster and Bren- nicke 1991), and Petunia (Hanson et al. 1993), rps7 in wheat (Zhuo and Bonen 1993), rpsl2 in wheat and maize (Gualberto et al. 1989), Oenothera (Schuster et al. 1990b), and ginseng (Kim et al. 1991), rpsl3 in tobacco and maize (Bland et al. 1986), wheat (Bonen 1987), Oenothera (Schuster and Brennicke 1987), and carrot (Wissinger et al. 1990), rpsl4 in broad bean (Wahleithner and Wolstenholme 1988), Oenothera (Schuster et al, 1990a) and Arabidopsis (Aubert et al. 1992), rpsl9 in Petunia (Conklin and Hanson 1991) and Oenothera (Schuster and. Brennicke 1991), and rpll6 in maize (Hunt and Newton 1991) and Petunia (Hanson et al. 1993). In some plant species, however, several of these genes ap- pear to be non-functional pseudogenes in mitochondria, because translational stop signals, frame shifts and 5' and/or 3' deletions disrupt the reading frames. The intact functional copies of these genes have presumably been transferred to the nucleus. In Oenothera, where only a transcribed, but incomplete pseudogene of rpsl2 is found in mitochondria, the functional gene has indeed been identified in the nucleus (Grohmann et al. 1992).

In Escherichia eoli, the ribosomal protein genes are organized in the $10, spc and ~ operons (Lindahl and Zengel 1986). The large ribosomal cluster in higher plant plastid DNAs appears to be derived from a prokaryotic ancestor with a related gene organization (Sugiura 1989). A similarly conserved gene order was found in Marchan- tia mitochondria, where fourteen ribosomal protein genes are arranged in two clusters (Oda et al. 1992). Only rudiments of this organization are present in higher plant mitochondria, while most of the ribosomal genes are

446

dispersed around the genome as a result of the high recombinational activity.

By cDNA analysis a new edited open reading frame (ORF) has been identified upstream of the gene coding for subunit 3 of the NADH dehydrogenase (nad3) in Oenothera mitochondria. As shown here, this ORF en- codes a protein corresponding to the prokaryotic ribo- somal protein L5.

Results and discussion

Organization of the rpl5 locus in Oenothera

In Oenothera mitochondria, the nad3 gene is located upstream of a rpsl2 pseudogene on a 3.5 kb HindIII restriction fragment. This gene locus is transcribed into a number of transcripts ranging in size from 1.2 to 7.5 kb.

Material and methods

DNA preparation and Southern analysis. Mitochondria of O. berteriana were isolated from tissue cultures by differ- ential centrifugation followed by Percoll gradient centri- fugation (Schuster et al. 1989). After DNase I digestion of the isolated mitochondria, the mtDNA was further purified by equilibrium density centrifugation in CsC1 gradients. Total mtDNA was digested with restriction enzymes, fractionated by electrophoresis in agarose gels and transferred to Biodyne A membranes (Pall). Hy- bridization analysis was carried out using standard procedures (Sambrook et al. 1989). After hybridization, blots were washed at 60 ° C in 0.1 × SSC, 0.1% SDS and autoradiographed at - 8 0 ° C.

PCR amplification and cDNA construction. Mitochon- drial RNA (mtRNA) was isolated from purified Oeno- thera mitochondria as described previously (Wissinger et al. 1991). After DNase I digestion the mtRNA was re- verse-transcribed using random hexamer primers, and the resulting first strand cDNA was used for the specific PCR amplifications. The PCR reaction mixtures con- tained 50 mM KC1, 1.5 mM MgC12, 10 mM TRIS-HC1 pH 8.3,500 ng of each primer, 0.05 mmol of each dNTP, 10 ng cDNA and 2.5 units of Taq polymerase (Boehrin- ger). PCR was performed on a Biomed cycler under the following conditions: 1 min at 95 ° C, 1 min at 55 ° C and 2 rain at 72 ° C. This cycle was repeated 30 times with a period of 10 rain at 72 ° C. PCR amplifications were done with the following primers: upstream of rpl5, 5"- AAGAGAGAAAAGTTTGG-3 ' ; downstream of rpl5, 5'-CAGTTCGGTCAAGATGC-3'; downstream of nad3, 5'-TTTTCCCTCTTACTAGTG-3'. The rpl5 am- plification product was digested with EcoRI and NsiI, the rpl5-nad3 product with EcoRI and SpeI, and both were cloned into Bluescript (Stratagene) vectors.

Sequence analysis. A dideoxy double-stranded sequenc- ing procedure for T7 DNA polymerase (Pharmacia) was used in sequence analysis with T3 and T7 primers. The genomic sequence presented was determined on both strands in overlapping subclones isolated from the origi- nal HindIII clone, rpl5 and rpl5-nad3 PCR-derived cDNA clones were sequenced using custom-made oligo- nucleotide primers.

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Fig. 1. Restriction map of the rpl5 locus in Oenothera berteriana mitochondria. The rpl5 gene is located upstream of the nad3 gene and the rpsl2 pseudogene on a 3.5 kb HindlIl fragment. Below this, map location and length of the cDNA clones analysed are shown as solid lines, cDNA products obtained by PCR amplification with the primers indicated by horizontal arrows are depicted as dotted lines. RNA editing sites identified by sequence comparison of the genomic and cDNA sequences are indicated by vertical arrows above the rpl5 and nad3 genes. The hybridization probe used in Southern experiments is shown as a hatched bar and covers rpl5 and upstream sequences

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Fig. 2. The rpl5 gene is present in a sing- le location in the mitochondrial genome of Oenothera berteriana. Southern hy- bridization with rpl5 and upstream sequences (indicated in Fig. 1) identifies a single genomic locus in total mtDNA digested with BamHI or HindlII. Sizes of length markers are given on the left in kb

1

61

121

181

241

288

333

378

423

468

513

558

603

648

693

738

783

828

886

946

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CCTGAACCACTCTAAGAAGAATTTTTAAATTGGTAATACACACTTGAAGTATAAGAAAAG

TGTTGGTGCAGAGATAACGTATATAGAATGGATTTTTTCTTTCTGATCACTAGCTGCCGG

ACCGCCCTCTCGATCAAACTATCAATTTCATAAGAGAGAAAAGT TTGGAATTCGACCTCC

CCGATCTACTTT TTTTTTTTAGTGGGAAACCCTGTCAATTGAATTTCTAAATAT TATACT

rpl5 ~ M F P L N F H Y E D V GAAAAATCGTAGTC ATG TTT CCA CTC AAT TTT CAT TAC GAA GAT GTC

CGT CAG GAT CTT TTG CTC AAA[C~G~]~AC[AAT GCC AAC GTT ATG

E V P G ~ C E I R I V P K T S GAA GTT CCT GGA T ~ TGT GAA ATC AGA ATA GTA CCA AAG ACA TCC

S T Y D F I I K N G K L A M E TCG ACC TAC GAT TTC ATA ATA AAA AAT GGA AAA TTG GCT ATG GAG

I L R G Q K F I Q T E R G S T ATT CTG CGG GGT CAG AAA TTC ATA CAG ACA GAA AGG GGT TCG ACA

G K S F R S N P F L E S N K D GGA AAG TCA TTT CGA TCC AAT CCA TTC TTG GAG TCA AAT AAA GAC

K G Y V S D L A R Q S I L R G AAA GGA TAT GTC AGT GAC CTA GCA CGA CAA AGT ATC CTC CGA GGG

o s v s CAT GGA ATG TCT AAT TTT ]T~G[GTC AGA ATC]T~G[ACT GTA ATG TCT

L L D S R V E I R E N S I Q F CTG TTA GAT TCT CGG GTC GAA ATA CGG GAA AAC TCA ATT CAA TTC

S M E T E F C E F S P E L E D TCG ATG GAA ACG GAG TTT TGC GAA TTC TCC CCG GAA CTG GAA GAT

H F E I F E H I R R F N V T I CAT TTC GAG ATC TTC GAA CAT ATT CGA AGG TTC AAT GTT ACT ATT

V T S A N T Q D E T TLAT P~L P~L W S GTC ACT TCG GCC AAC ACC CAA GAT GAG ACT CCA C/:G TGG AGC

G F L Q K D E G E S F K W K T GGC TTT TTG CAA AAA GAT GAG GGG GAA AGT TTC AAA TGG AAA ACT

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CCT TAA AGTCAAATGCATCTTGACCGAACTGAGAGAAAACCTTGTTTTGATAGGATTT

CTTCTTCTGTGACTTTATATTTCTAAAATGAACGCCCATTTTTCCTGATGCTAAGTCCGC

CGTAGCCCTTCTAGGGATACATCTTCTAGCACTAAAGTGCAAGATTCAACAACTATGGAA

TGAATCTAGTTGCGGGATGGGTTTTTCCGGTATGCCGCTCCGCGAGCAAGGAGCGAAAGA

nad3 ~ M ~-~ E F A P I C I S ACAAAGTGGGCTGTGGTG ATG[T~A] GAA TTT GCA CCT ATT TGT ATC TCT

447

Fig. 3. Nucleotide sequence of the rpl5 locus and flanking regions in Oenothera. RNA edit- ing sites are underlined and the altered amino acids are boxed. The amino acid sequence de- duced from the DNA is shown in the single letter code. The EMBL Data Library acces- sion number for this sequence is X69553

Analysis of cDNA clones covering these genes revealed an additional ORF upstream of the nad3 gene (Fig. 1). Hybridization with this ORF and its flanking regions identified single restriction fragments for HindIII (3.5 kb) and BamHI (7.5 kb) indicating that this ORF occurs only once in the Oenothera mitochondrial genome (Fig. 2). In wheat and maize mitochondria the rps12 and had3 genes are also cotranscribed (Gualberto et al. 1988), but the upstream sequence differs completely from that in Oenothera.

Analysis of the rpl5-nad3 transcription unit

Sequence analysis of the 3.5 kb HindIII clone from Oenothera berteriana revealed in this upstream ORF the

high percentage of T in third codon positions commonly found in plant mitochondrial genes (Fig. 3). The open reading frame of 576 bp in length could code for a pro- tein of 192 amino acids. Comparison of the deduced polypeptide using the FASTA program (Pearson and Lipman 1988) showed significant similarity with rpl5 sequences of Marchantia (Oda et al. 1992), E. coli and Bacillus stearothermophilus (Kimura and Kimura 1987). The deduced rpl5 polypeptide sequence from Oenothera shares 61% identity with the Marchantia protein, and about 20 % with the respective E. coli and Bacillus homo- logues (Fig. 4). It has been shown that ribosomal protein L5 (RPL5) can bind specifically to 5S rRNA, and in re- constitution experiments the RPL5 proteins from E. coli and B. stearothermophilus are interchangeable (Kimura and Kimura 1987). The low degree of conservation of the

448

Oenothera gen.

Oenothera cDNA Marchantia E. coli Bacillus st.

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Oe cDNA March. E. coli B. st.

S S I l 150

. . . . . |L S E EIErCEFSmLE LV~II ..... mFY~YP~KUQK~L~A~SLLRLF~IBQ~ LILAVPRIRDFRGLSAKSFDGRGDYR~VREQZIFII-|-YDK LIS~SLPRVRDFRGVSKKAFDGRGNYTLGIKEQLIF'I#~Z-~-YDK

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VORV LDIII T S L FPFRK* VNKV~GMD I V~DE~ARE[ILAL LGMPFQK *

Fig. 4. Alignment of L5 ribosomal protein amino acid sequences from Oenothera berteriana mitochondria, Marchantia poIymorpha mitochondria (Oda et al. 1992), Escherichia coli and Bacillus stearothermophilus (Kimura and Kimura 1987). Amino acids al- tered by R N A editing are indicated. Positions identical to the Oenothera RPL5 amino acid sequence are shown in inverse type

Oenothera and Marchantia RPL5 proteins relative to their prokaryotic counterparts may be due to adaptation to different sequences of the 5S rRNA.

In Marchantia mitochondria, the rpl5 gene is part of a ribosomal cistron containing 12 genes, which is only partially conserved with three genes (rpsl9, rps3, and rpll6) in Oenothera mitochondria (Schuster and Bren- nicke 1991). The rpl5 gene has been translocated to a dif- ferent genomic location and is now part of a transcrip- tional unit with the nad3 gene in Oenothera. Cotranscrip- tion of the rpl5 and nad3 genes is confirmed by both, cDNA cloning (Fig. 1) and Northern blot analyses (Schuster et al. 1990b).

RNA editin 9 of the rpl5 readin9 frame

To identify the extent of RNA editing in the rpl5-nad3 transcripts, sequences of cDNA clones and PCR-derived cDNA were compared to the genomically encoded in- formation. In the translated region of the rpl5 gene, nine DNA-encoded cytidines are replaced by thymidines in the cDNA. Eight of these edits are non-silent and change the amino acid sequence to a polypeptide showing greater conservation relative to L5 proteins of other plant and non-plant species. Seven of these eight non- silent edits change the respective triplets to leucine co- dons, the most frequent amino acid change introduced by the editing process. The single silent editing site is edited at a much lower frequency than the other eight sites. Only 10% of the cDNA clones analysed show this transition. In the nad3 coding region eleven C to U edits are frequently observed altered, while two additional non-silent editing sites are modified to a much lower degree (Schuster et al. 1990b). No evidence for RNA editing has been observed in the transcribed spacer re- gions separating the two genes.

rpl5 sequences in other plant mitochondria

At least parts of the ORF coding for rpl5 are also present in mitochondria of other higher plants. In Arabidopsis thaliana mitochondria, sequences homologous to the C- terminal part of the rpl5 of Oenothera are located up- stream of a rps14 pseudogone which is transcribed and edited (Aubert et al. 1992). This gene arrangement seems also to be conserved in Vicia faba (Wahleithner and Wolstenholme 1988). In this plant, only 60 nucleotides of rpl5 are present, indicating this locus to be a pseudo- gene. In Oenothera the ribosomal cistron is differently organized because rearrangements have moved virtually all genes to other genomic locations. The original site of the rpsl9, rps3, rp116, and rpl5 genes is now occupied by an ORF (off206) coding for a protein that is prob- ably involved in cytochrome c biogenesis (W. Schuster and A. Brennicke, in preparation). This translocation re- sults in the rpl2 gene now being separated from rps14 by the inserted orf206. Another rearrangement translo- cated the rpl5 gene to its present location upstream of nad3 and created a new transcriptional unit.

Acknowledgements. I would very much like to thank Dr. A. Bren- nicke for his interest and encouragement. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Bun- desministerium fi.ir Forschung und Technologie.

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