Curt Genet (1989) 15:187-192 Current Genetics © Springer-Verlag 1989

Conserved sequence elements at putative processing sites in plant mitochondria

Wolfgang Schuster and Axel Brennicke Institut ftir Genbiologische Forschung, Ihnestrasse 63, D-1000 Berlin 33

Summary. About 50 nucleotides are conserved in two distinct sequence elements at the 5' termini of abundant short transcripts from plant mitochondrial genes with complex transcription patterns. These sequence motifs are found in both structural RNA and protein-coding precursor transcripts, for example, upstream of the mature 26S rRNA and the shortest transcript of the ATPase subunit 9 genes in different plant species. The high degree of sequence similaritiy in these plant mitochondrial loci suggests a common functional significance in 5' processing site determination and selection.

Key words: Processing sites - Plant mitochondria - Oenothera -ATPase subunit 9 gene - 26S rRNA transcripts

Introduction

Many of the plant mitochondrial genes identified to date are transcribed as monocistronic transcription units into one single mRNA species for each gene. Several genes, however, are consistently expressed in a complex array of mRNAs in the various plant species that have been investigated in this respect. Analysis of the initiation and processing events generating these complex transcript patterns is a prerequisite to deter- mining their functional significance.

One of the genes with multiple trancripts in diffe- rent plant species encodes subunit 9 of the ATPase complex (atp9; Dewey et al. 1985a, b; Bland et al. 1986: Rothenberg and Hanson 1987). This article presents the results of our investigations on the 5' and 3' mRNA termini of both this gene and the large riboso-

Offprint requests to: A. Brennicke

mal RNA in mitochondria of Oenothera; these sequen- ces are compared with the 5' terminal sequences of other plant mitochondrial transcripts. The strong sequence homology observed between the 5' termini of the mature, processed large ribosomal RNA and the short atp9 transcripts in different plant species suggest that this conserved sequence is involved in guiding a specific processing activity.

Materials and methods

The methods employed in identification, sequencing, and transcript analyses of the atp9 and 26S rRNA genes in Oenothera mitochondria have been described previously (Hiesel and Brennicke 1983; Manna and Brennicke 1985; Schuster and Brennicke 1987). The new sequences reported in this communication (Fig. 1) have been determined overlapping at least twice on both strands by controlled chemical modification (Maxam and Gilbert 1980).

Results and discussion

Analysis of the atp9 gene

The gene encoding subunit 9 of the ATPase is located in the mitochondrial genome of Oenothera upstream of a sequence of chloroplast origin with part of the 23S rRNA and the 5.8S rRNA (Schuster and Brennicke 1987). The highly hydrophobic membrane-internal subunit 9 of the ATPase complex is specified by a continuous reading frame of 234 nucleotides in Oeno- thera (Fig. 1). Of the 78 codons 33.3% contain a T in the third position (31% A), a characteristic of genuine plant mitochondrial genes. The deduced polypeptide is one amino acid longer than the respective tobacco (Dewey et al. 1985b) and Petunia (Rothenberg and Hanson 1987) proteins and contains four extra amino acids at the carboxy terminus in comparison with the maize and pea polypeptides. Internal similarity

188

S~p I -517 T~TA2ACTA~ATrCTATAAAAGATA~AAATAA~AAGATATATACATATATA~T~AC~7~CATAAGAAGA~AGTrGACCTTTCCT~

-417 CTGTTGCCC~CAACC~AATCGGAATrAG6"rAAGA AC~ AAAC~ATATGAAATGAATI~rAT/-rGAATATC~ATr t:ri~rrl-rlL~AGA~

-217 T~ATCGTGAATAAAAA~GCGT~GGGAGACTrGAAAACTrTGATATGAGAGATrGCCTTACAGL.rrxL.rrr~AT~G.~rrrrrrr~TCGAAAGCAGAAAG

-I 17 TCCTGAC, GCGGACATCTUrGTGATGCCGAAC~ACGAACTC~GAAGG AAAATGAAAGCCCACATI~CAAAG AAA0~A~AG~ATAAGAACG~ A

M L E G A K S M C S O A A T I A L A G A -17 ACAA~'fGATCAATTATr ATG "IrA GAG GGT GCA AAA TCA ATG GGG TCG GGA GCT GCT ACA ATr GCT CTA GCG GGA GCT

A I G I G N V F S S L I H S V A R N P S L A l Q L 61 GUt ATC GGT ATr GGA AAC GTC TI~ AGT TCT Tr0 ATT CAT TCC ~A GCC AGA AAT CCA TCA "IrA cur AAA CAA TrG

F G Y A I L 0 F A L T E A I A L F A P M M h F L I 136 TIT GGT TAT GCC ATr TrG GGC TIT GCT CTA ACC GAA GCT ATT GCA TrG TIT GCC CCA ATG ATG GCC TIT TrA ATC

L F V F R $ V I[ * 211 TrA TIT GTA TrC CGA TCG ~ AAA TAG GGTITCAGGCTCATAAAGCAAGCACCATCCACTI~GGATGCGAATGGCTCAC, CAGGGGTACTCG

302 TC~ACCACCCTC~AGAATCI~CCGTCAGCATGGCI~ ACTCGGAAC~CATCCAC~CA'I'GTCA~GAA~G

Ha., HI 402 AATCTC-GGACGAOTC, CAGGATCC-I~I-rlTITrrrr rIA2~I'GACGAAAGAAG'I~ACTC~AGAGGCC~ATATGTA~AAAA~AA AC~

Fig. 1. Localization of the short transcript termini in the nucleotide sequence of the ATPase subunit 9 gene (atp9) in Oenothera mitochondria. Arrows indicate 5' termini of transcripts as determined by nuclease protection experiments. The different sizes of these arrows reflect the relative abundance of each transcript. A and B denote sites that are experimentally defined in Fig, 2

amounts to approximately 90 % between the different- higher plant genes (and deduced proteins) and to 55 % between the Oenothera and the nuclear encoded Neuro- spora (Sebald et al. 1980) polypeptides. Two large hydrophobic regions between amino acids 8-32 and 45-72 are responsible for the hydrophobic nature of this protein. Sequences outside of the reading frame diverge between different higher plant species, but the transcript pattern seems to be conserved.

Transcription of atp9

The Oenothera atp9 locus has homologous sequences in a number of transcript species of various sizes (data not shown) that are very similar to those found in the atp9 genes in other plant mitochondria (Dewey et al. 1985b; Bland et al. 1986; Rothenberg and Hanson 1987). Not all transcripts detected with probes from the atp9 coding region must necessarily be transcripts from this locus in all plant species. For example, in maize mitochondrial RNA(mtRNA), such an atp9 probe also detects the coxltranscripts due to an approximately 80 nucleotide sequence duplicated from the internal atp9 region that overlaps the last few codons of the cox! reading frame.

Nuclease protection experiments give an impression of relative transcript abundances at different 5' termini (Fig. 2). Most of the atp9 mRNAs begin 284 nucleoti- des upstream of the initiation codon (Site A in Figs. 1 and 2). The next upstream 5' mRNA termini are located 456 nucleotides 5' of the open reading frame (ORF) (Site B in Figs. 1 and 2). At least one other terminus of larger transcripts is detected further up- stream of this site (data not shown). Several of the large precursors of the maize atp9 gene appear to derive from separate initiation events (Mulligan et al. 1988). The relative mRNA stoichiometries of the Oenothera atp9 locus are very similar to the transcript pattern of the Petunia atp9, where abundance levels have been rough- ly estimated to be 100: 5:1 between the shortest and the progressively longer transcripts (Rothenberg and Han- son 1987).

The major 3' terminus of the Oenothera atp9 transcript population is located approximately 250 nucleotides downstream from the termination codon (data not shown).

For comparison with the 5'-atp9 mRNA termini we also analyzed the Y-transcript termini of the 26S rRNA locus, the only other monocistronic gene with multiple transcripts so far identified in Oenothera mitochon- dria.

189

Fig. 2A, B. Nuclease protection experiments to determine the 5' termini of the atp9 transcripts. A shows the relative stoichiometries of the mRNA species in two different exposures, mtRNA protects specific DNA sequences (lane $1) in comparison to the degradation observed with yeast tRNA in the control reaction (Ko). The scattered 5' termini at site A are resolved at the nucleotide level in the sequencing gel shown in B Lanes I and 2 show S l nuclease protection reactions with yeast tRNA (lane 1) and mtRNA (lane 2). Tracks 3, 4, 5 and 6 show the sequencing ladder of this region in the order G, G+A, C+T and C

Transcription of the 26S rRNA gene

Nuclease protection experiments f rom within the ma- ture 26S rRNA gene into the upstream region identified a precursor 20-30 nucleotides longer than the mature 26S rRNA (Fig. 3) as well as other, much larger precursor RNAs of low abundance (not shown). In vitro capping experiments have shown that transcrip- tion of the 26S rRNA in maize mitochondria is initiated some 180 nucleotides upstream of the mature rRNA, which appears to arise solely f rom processing events (Mulligan et al. 1988).

The mature 26S rRNA terminus in Oenothera maps to the same nucleotides that have been identified in

other experimentally determined 26S rRNA termini in wheat (Falconet et al. 1988) and maize mitochondria (Mulligan et al. 1988). The mature 26S rRNA thus appears to be shorter at the 5' terminus than previously suggested by comparison experiments with the respec- tive E. coli sequence (Dale et al. 1984; Manna and Brennicke 1985) and appears to begin just before the first paired region in the rRNA molecule (Manna and Brennicke 1985).

Homologies at putative processing sites

Comparison of the nucleotide sequences around the shortest transcripts of the ATPase subunit 9 gene and

190

Eco R ~"

,I NcoI

26 S r DNA

NcoZ (670 nf]

[ ~> pre 26S rRNA

A I ~> mo.l~ure 26S rRNA

Oe 265 rRNA pre 26S rRNA

GGCTTGGTGTTCAGTGTACCAAACCCAATAA AGTGTAGCCTATCCGAAGCAAGCCTTATTTATAAGCTTAG~--'TTAG

GGGGGTACAAGATC~TGCA

D ma~ure 265 rRNA

191

Oenothera atp9

Oenothera rrn26

TTTTTCT 1TCGAGATTGGCTTGGTGTTCAATGTGCC

;;;C;TO::::::::::::::::::::::::::::: CCAAGTGA -268

GGATGGGT 14

!!!!{

...... TTA[GTACAAGATCGAAAAGAATGCATT

GCTCATGTT CGACGCTATCGAAAATCATGCATT GCTCATGTT CGACGCTATCGAAAATCATGCATT ACTCATGTT CACGTTCTACAAATCTAAGGGAGT GCT .... TG TCTAGCCT ......... ATGCTTT GCT .... TG TCTAGCCT ......... ATGCTTT GCT .... TG TGACGCCT ......... ATGCTTT GCTCTTTTT ATATATGAAATTACTTCGTCCTTT CTACCGCTC ATNNNNGATTTCGTTGGGTAGAAC GAAATGGGT GAAGTCTCTTTCAAGAAAAGAGCA

Maize rrn26 TGTCAAA TTGAGATTGTGTGGGTGTTCAGTCTACC GGATGGGT 21 Wheat rrn26 TGTCAAA TCGAGATTGTGTGGGTGT ........ CC GGATGGAT 19 Maize 5.27 kb TGTCAAA TAGAGATTGTGTGGGTGTTCAGTCTACC GCTCCTAG 842 Petunia atp9-1 TTATTTT TCGAGATTGGGTTGGTGTTCAGTGTACC GCATGAAC -107 Petunia atp9-2 TTTTGCG TCGAGATTGGGTTGGTGTTCAGTGTACC GCATGAAC -107 Tobacco atp9 TTCTTTC TCACGATTGGGTTGGTGTTCAGTGTACC GCATGAAC -106 Tobacco atp6 TAGTTCT TTGATATTGGGTTCGTGTTCAGTGTACC TTTTTAGC -141 Maize atp6 TGTCAAA TCGAGATTGTGTGGGTGTTCAGTCTACT CAAGTCTC -398 Maize T-urfl3 TGTCAAA TCGAGATTGTGTGGGTGTTCAGT-TCAT GAGGGAGC - 25

Fig. 4. Sequences around the 5' termini of the shortest transcripts of the atp9 gene and the mature 26S rRNA of Oenothera mitochondria are aligned with analogous regions from several other genes of different plant species. Nucleotides identical between the two Oenothera genes are indicated with asterisks, the two conserved regions are boxed, and the major 5'-processed RNA termini are indicated by dashes. These RNA termini have been experimentally determined in the Oenothera 26S and atp9, maize and wheat 26S rRNA, and Petunia atp9-1 and atp9-2 genes. Alignments with the first conserved region include the homologies previously observed in the tobacco atp6 5' flanking region (Bland et al. 1987). Abbreviations: atp6 and atp9, genes encoding subunits 6 and 9 of the ATPase; rrn26, genes encoding ribosomal 26S RNA; maize 5.27 kb is a direct repeat in the maize mitochondrial genome; T-urf13 encodes a mosaic gene in the T-cytoplasm of maize (Lonsdale and Leaver 1988). Data for maize, wheat, Petunia and tobacco are taken from the following references: maize rrn26 (Dale et al. 1984; Mulligan et al. 1988); wheat rrn26 (Falconet et al. 1988); maize 5.27 kb (Houchins et al. 1986); Petunia atp9-1 and atp9-2 (Rothenberg and Hanson 1987); tobacco atp9 (Bland et al. 1986); tobacco atp6 (Bland et al. 1987); maize atp6 (sequences upstream as far as nucleotide -423 in Dewey et al. 1985a; further upstream from - 428 to --473 in Stamper et al. 1987); Nindicates a nucleotide with unpublished identity); T-urf13 (Dewey et al. 1986)

the mature 26S r R N A in Oenothera reveals a high degree of specific sequence conservat ion in this region (Fig. 4). These sequence similarities extend to a number o f other complexly transcribed mi tochondr ia l genes f rom different plant species.

Two distinct blocks o f h o m o l o g y can be discerned in the approximate ly 50- to 70-nucleotide-large region of sequence similarity. The division into two separate sequence elements is suggested by the variable distance between the two motifs, for example, the insertion o f some 60 nucleotides between these blocks in the Oenothera 26S r R N A locus. The resulting exclusion of the ups t ream element f rom the short 26S r R N A precursor might lower the efficiency of the processing react ion in this molecule. Such a diminished processing rate could explain the presence o f detectable amounts o f this precursor in Oenothera mitochondr ia and the absence o f similar, detectable amounts o f larger 26S r R N A precursors in the steady state m t R N A of wheat (Falconet et al. 1988).

A separate funct ion o f the two sequence elements is fur ther suggested by the divergent degree o f conserva- t ion o f the two regions in different mi tochondr ia l loci. The regions o f h o m o l o g y with the distal element, for

example, are easily detected upst ream of atp6 genes and have therefore been included in Fig. 4 in an al ignment similar to the previously reported h o m o l o g y between these genes (Bland et al. 1987). The locat ion o f the corresponding processing site and the second conserved element in these atp6 loci, however, cannot be defined th rough h o m o l o g y searches alone, but requires experimental determinat ion o f these 5'- m R N A termini.

The proximal sequence block actually covers the putative processing site, with the first four nucleotides (5 ' -ATGC-3 ' ) o f the processed R N A being highly conserved. The exact processing sites appear to vary within a few nucleotides, since a scattering o f protected termini is observed in the nuclease protect ion experi- ments. This could, o f course, alternatively be artifactu- al in the S 1 reaction, and therefore needs to be investigated with different methodologies.

These two conserved elements are distinctly diffe- rent f rom the conserved sequence block at the 5' termini o f singular transcripts that has been proposed to be par t o f the p romote r recognit ion site in higher plant mitochondria . These putat ive p romote r sequen- ces are found at the m R N A 5' ends o f genes with single

Fig. 3A-D. Transcript 5' termini of the 26S rRNA locus in Oenothera mitochondria. A 5' termini of mature and precursor 26S rRNA molecules were determined by sizing the nucleotide chain in the labeled (asterisk) NcoI/EcoRV fragment protected from nuclease digestion. B The stoichiometries of the short precursor (pre) molecules and the mature 26S rRNA on a 4% polyacrylamide gel. C Accurate nucleotide assignment of the RNA protected antisense DNA strand along the sequence ladder of the same restriction fragment. Long exposure shows the terminal nucleotides protected by the precursor transcript in the lane marked S 1. Yeast tRNA (20 Ixg/lll) was used instead of mtRNA in the control reaction (K0). D Nucleotide sequence of the region 5' to the mature 26S rRNA. The two nucleotide positions protected by the precursor are enclosed by one bracket (pre 26S rRNA); the other bracket delimits the range of nucleotides observed for the mature 26S rRNA. The distal precursor signal indicates a leader sequence of 20-30 nucleotides to the 5' terminus of the mature 26S rRNA

192

(or few) transcripts e.g., cox! in maize (Isaac et al. 1985); coxllin Oenothera (Hiesel and Brennicke 1985); atp9 in Petunia (Young and Hanson 1987); cox! and coxllIin Oenothera (Hiesel et al. 1987) and the termini of the rRNA precursors in Oenothera mitochondria (summarized in Schuster et al. 1987).

Origin of the putative processing signals

Both the unique identity and the location of the two conserved sequences suggest their involvement in the highly efficient processing events required for matura- tion of sufficient quantities of the r ibosomal RNAs. A necessity for rapid processing would explain the high degree of sequence conservation of the 26S r ibosomal RNA matura t ion sites between different higher plant species. The homologous regions at the transcript termini of atp9 mRNAs seem to have the same functional effect, since only comparat ively low amounts of larger precursors are observed in the steady-state m R N A populat ion of these loci.

These sequences similarities might have been requi- red by the presence of only a single processing specifici- ty in plant mitochondria for the matura t ion of the rRNAs. This enzymatic activity would then be employ- ed to reduce atp9 transcripts to a translatable size requiring, for example, the conserved sequence ele- ments. Alternatively, regulatory processes could be involved in the processing steps of transcript precur- sors. Such a posttranscriptional regulation would explain the high degree of conservation in the complex transcript patterns for only some specifically selected genes in many different plant species. Functional pressure might have maintained the conserved proces- sing sequence elements in their relative locations during species evolution, after they had been duplica- ted and relocated by the frequent mitochondrial se- quence arrangement events.

Some of the homologous regions are actually derived f rom duplication events of the r ibosomal processing sites, as described for the urf13-T (Dewey et al. 1986) and the 5.27 kb (Houchins et al. 1986) repeat homologies in maize. Such a dispersion of the functio- nal signal sequences into different genomic loci via duplication in plant mitochondria has been observed for transcript termination and /or 3'-end stabilization (Schuster et al. 1986) and promoter regions (Dewey et al. 1986; Young and Hanson 1987; Hiesel et al. 1987).

The expansive regions of homology with the riboso- mal RNA processing sites in other genomic locations could similarily have been derived and successively degenerated f rom duplication events of the respective rRNA regions in evolutionary time. A functional selection pressure on efficient splicing by the rRNA

specific processing activity could have conserved the essential core sequences. This putative functional con- straint on these sequences now needs to be experimen- tally tested with in vitro assays of plant mitochondrial extracts to obtain direct evidence for the active involve- ment of these conserved sequence blocks in processing events to substantiate the so far only circumstantial evidence.

Acknowledgements. We are grateful to Prof. C. S. Levings III for the maize atp9 probe and Prof. C. J. Leaver F.R.S. for his critical help with the manuscript. The experiments described here were done in the Lehrstuhl Ftir Spezielle Botanik (Universit~it Tiibingen) of Prof. E Oberwinkler, whom we thank for his kind support. This work was financed by the Deutsche Forschungsgemeinschaft and a Heisenberg fellowship.

References

Bland MM, Levings CS III, Matzinger DF (1986) Mol Gen Genet 204:8-16

Bland MM, Levings CS III, Matzinger DF (1987) Curr Genet 12:475-481

Dale RMK, Mendu N, Ginsburg H, Kridl JC (1984) Plasmid 11 : 141- 150

Dewey RE, Levings CS III, Timothy DH (1985a) Plant Physiol 79:914-919

Dewey RE, Schuster AM, Levings CS III, Timothy DH (1985b) Proc Natl Acad Sci USA 82:1015-1019

Dewey RE, Levings CS III, Timothy DH (1986) Cell 44:439-449 Falconet D, Sevignac M, Querier F (1988) Curr Genet 13:75-82 Hiesel R, Brennicke A (1983) EMBO J 2:2171-2178 I-Iiesel R, Brennicke A (1985) FEBS Lett 193:164-168 Hiesel R, Schobel W, Schuster W, Brennicke A (1987) EMBO J 6:29-

34 Houchins JP, Ginsburg H, Rohrbaugh M, Dale RMK, Schardl CL,

Hodge TP, Lonsdale DM (1986) EMBO J 5:2781-2788 Isaac PG, Jones V, Leaver CJ (1985) EMBO J 4:1617-1623 Lonsdale DM, Leaver CJ (1988) Plant Mol Biol Rep 6:14-21 Manna E, Brermicke A (1985) Curr Genet 9:505-515 Maxam A, Gilbert W (1980) Methods Enzymo165:499-560 Mulligan RM, Maloney AP, Walbot V (1988) Mol Gen Genet

211 : 373-380 Rothenberg M, Hanson MR (1987) Mol Gen Genet 209:21-27 Schuster W, Brennicke A (1987) Mol Gen Genet 210:44-51 Schuster W, Hiesel R, Isaac PG, Leaver C J, Brennicke A (1986)

Nucleic Acids Res 14:5943-5954 Schuster W, Hiesel R, Wissinger B, Schobel W, Brennicke A (1987)

In: von Wettstein D, Chua N-H (eds) Plant molecular biology. NATO ASI series vol 140. Plenum Press, New York London, pp 115-126

Sebald W, Hoppe J, Wachter E (1980) Proc Natl Acad Sci USA 77:785-789

Stamper SE, Dewey RE, Bland MM, Levings CS III (1987) Curt Genet 12:457-463

Young EG, Hanson MR (1987) Cell 50:41-49

Communicated by C. J. Leaver

Received September 2l, 1988/January 4, 1989