mlOMMENT RNAediting fixes problems inplant mitochondrial transcripts \ilRGINIA WALBOT DEINHTMENT OF BIOLOGICAL SCIENCES, STANF~KU UWERSITY. ST~TFORD, CA 943055020, USA
RNA editing in transcripts of higher plant mitochondria increases the similarity of the predicted proteins with products of yeast and mammalian genes. makes the predicted proteins nearly identical in different plant species. cam create start and stop codons in the open reading frames, and allows translation using universal codon rules. Editing is extensive, altering up to 13.50/o of the codons, and is concentrated in the coding regions. Many incompletely edited transcripts can be recovered as cDNAs from plant suggesting that mitochondria, protein polymorphism could exist. Editing is, however, more complete in spliced than in unspliced transcripts, and polysome-associated mRNAs are edited ‘to a much greater extent than the tOhI transcript pool.
Complexities of plant mitochondrial transcription The plant mitochondrial genome, ranging in complexity from about 200 to 2500 kb and containing ciris cular and linear molecules, almost terra incognita compared with the smaller mitochondrial genomes of fungi and animals’. Plant mitochondrial transcripts are also very complex. On northern blots, multiple transcripts (>lO per geue) are the rule rather than the exception in many species. In maize. the multiple tr:mscripty for XVeral genes originate from independent initiation e\renta at promoters from 100 bases to sc\cr:ti kh upstream of the coding region of a gene’. In addition to genonie and transcript complexity. codon usage in higher plant mitochondria seemed to present confusing esceptions to princ$ple\ established in the other kingdoms. The initial reports on RNA editing resolved two of the long-.standing difficulties in understanding J’lant mitochonclI-ial gene .structure. First. a proposed exception to uni\.ersat codon usage i\ unnt-ctL\\:ir!
At
positions of conserved tryptophan codons in mitochondrial genes of fungi. mammals and many plants, some exceptional plant genes had a CGG rather than a UGG codon: consequently. both IIGG and CGG were proposed to encode tryptophanl. This proposal created a second problem, however. as CGG was found at conserved Arg positions. suggesting that CGG could encode either Arg or Trp. With RN.4 editing. C+C con\ vrsion restores IJGG codons in the conserved tryptophan positions, making codon usage in higher plants consistent with the universal codej-5. Second, although plant mitochondrial genes of closely related species are nearly identical, radical amino acid substitutions were proposed from the deduced protein sequences. For example, the cytochrome oxidase subunit 2 (~0x2) genes of wheat and Inaize shon 98.90/o nucleotide identity. Hut the three differences in the 260 residue predicted amino acid sequence in\,olve radical replacements (maize--+wheat): Glu(GAA)+Ser(TCA), I’ro(CCA)--+Leu(CTT), and Cys(TGT)-+Arg(CGT). The apparent loss of the Cys was predicted to affect cytochrome oxidase function as this site is involved in binding a catalyticall)
essential copper ion 1, .4gain. RNA editing comes to the rescue, 21sthe Arg(CGU) codon is edited to Cys(UGU) in the wheat message.
Extensive editing in plant mitochondrial transcripts More news on RNA editing is now available from the Fourth International Workshop on Plant Mitochondria held on 23-2: September 1990 and hosted by Cornell L’niversity and the RO)XX Thompson Institute. By direct RYA sequencing or sequencing of uncloned cDNAs, transcripts representing about 50 kb of genes have been compared with genomic DNA. In other studies, multiple cloned cI)NAs have been sequenced to determine the distribution of edited sites within individual mRNAs. These studies hdVt! utilized primarily two monocots (wheat. maize) and t\vo dicots (Oenoth~ra, RWr7ia). The two largest data sets are sunlmarized in Tables 1 and 2. Important conclusiona from thr (Iat: availahte thus far include: ( 1) i\ll protein-coding transcripts examined to date are edited (2) The extent of editing varies Lvidely from two to 23 edits pc transcript. (3) The impact of editing can he extensive: in the r?nt/.3 transcript of wheat. 13.7X of the amino acid
TABLE1. Editing in wheat mitochondrial transcriptsa _ .~_______
Editing sites thneb
c+u
u+K
Altered amino acids Number
12
1
12
21 19
0 0
tpsl2
6
0
or-1 56
4
0
16 18 6 3
cox3 na& nad4
%
4.5 13.5 3.8 3.8 1.9
“The data (supplied by J-M. Grienenberger, CNRS, Strasbourg) refer to sites found to be edited in at least one cDNA; editing is not 100% efficient. “cox, cytochrome oxidase. subunit by number; nad, NADH oxidoreductase, subunit by number; rps. small ribosomdl subunit protein, number indicates approximate size in kDa; orj open reading frame.
fIG
T‘FIIRI,\RY
1991 \-()I. 7 \O. 1
mlOMMENT residues predicted from the DNA sequence are altered in the mRNA (Table 1). In the maize ~0~2 transcript, one codon receives two edits: CCA+UAA (KM. Mulligan. University of California, Irvine). (4) There is a strong bias (87% in \\hcat. El”,, in Oerzotheva) for editing that alters the amino acid specified by the codon. (5) In wheat all of the known editing sites are within the coding region. but in Oetzothera and maize. edited sites in Lhe 5’ and .?’ unlranslated region have been found. (6) Editing can also occur in structural RNA: the 26s rRNA of Oenothera has two edited sites. (7) Nearly all editing detected so far involves C--+(i changes (62 out of 63 in wheat: 118 out of 121 in Oenothera), but LJ+C edits have also been detected”. (8) Editing improv-es the degree of similarity at the level of predicted amino acid sequence for individual proteins among the higher plants as well as in comparisons between plants, fungi and animals.
TABLE2.
Gem? atp1
atpG up9 cob cox2 co3E3 nadl exon exon exon exon
A
B C D
exon E Maturase
nad3 rpsl4
orf
OrJB 1% rRNA 5S rRNA 26s rRNA
Codiig region length and transcript recognition features
For some plant mitochondrial genes, estimation of expected protein size has been problematical. Despite nucleotide similarity in the amino-terminal coding region. an ATG could be missing from the ‘obvious’ placct in t!~c DNA sequence as parison with
deduced
from
com-
other genes. At the carboxy terminus. predicted plant were proteins often virtual15 Identical through the region correspending to the cognate fungal and animal coding regions. but then the plant open reading frames diverged extensively and continued for variable distances, Such discrepancies in trdnSlational start and stop sites have now been resolved for several genes. For example, the ATG start codon for rzadl in wheat is created by RNA editing (ACG+AUG) (L. Honen, UniVet’Sity Of Ottawd). For the atp9 gene of wheat, CGG+ UGG editing creates a stop codon at apparent codon position 75. resulting in a 74 amino acid protein. Editing at RNA editing in Oemtbera position 75 also occurs in tl%UlSCtiptia the other plant species, making the predicted proNo. of Editingsites teins the same length. c-m u-6 altered AA@ Future studies may also indicate a role for editing 4 0 2 outside the coding region: 12 0 12 two possibilities would be 4 0 4 to increase the match to the 13 1 12 consensus sequence for 22 1 17 4 0 4 40s ribosome interaction or to alter intron splicing 4 0 4 kinetics. 1 0 0 9 0 6 1 11 4 16 2 5 7 0 0 1
0 0 0 o 0 0 0 0 0 1
I 8 4 13 2 4 5 NA NA NA
The data (supplied by W. Schuster and A. Brennicke, Institut fiir Genbiologische Forschung, Berlin) refer to sites found to be edited in at least one cDNA. bAbbreviations as for Table 1 plus cob, apocytochrome h; a proposed maturase gene is encoded within nadl. u
CAAS,amino acids.
Editing pattern Iri m(Jst tr~li~%XiptS, the
clislritW&)n ot‘ editing \itY\ I4 random within the c,oding region There is. however. a highly cIu.4tered pattern of edited \ites in wheat rrad4: this primary transcript has three axons of similar size. hut 1’ out of 19 editing sites are in the first exon. and just tn.0 are in cxon 3. 15 there an or&r to the editing process? From the first reports of RNA editing rn plant mltochondrial transcripts. it 5vas clear that although the majority of
potentially edited sites are edited, most mRNAs are incompietel) edited.+‘. By sequencing multiple cDNAs containing pre-edited sites. an order to RNA editing might be deduced. In plants such analyses have so far failed to elucidate any particular order to RNA editing. The available data suggest that at best some regions of transcripts are edited more rapidly or efficiently than others, but there is no apparent i’+_S’ or 3’%5’ order to the process.
Does editing produce fully edited mRNA? Among eight cDNAs of uad-3 from Oenothem, each transcript had at least two unedited sites (at random positions, out of a total of 16). The persistence of pre-edited transcripts and the observation that most edits alter a codon opens up the possibility that protein isoforms are encoded by the diverse transcripts from a single plant mitochondrial gene. There is experimental evidence suggesting, however, that translatable mRNAs are more completely edited than the total transcript pool. First. for transcripts undergoing both editing and intron splicing. editing is more complete in the spliced than unspliced molecules (C. Sutton and IM. Hanson, Cornell University). Second, polysomal RNA is almost completely edited (Mulligan). Thus, the functional mRNAs in plant mitochondria may each encode a single major protein form. Evaluation of the efficiency 01 editing and the uniformity of protein products will require analysis of mitochondrial proteins. Only for n.hcar tit]).9 ha\.c thrcr rclrting site5 ht,W ~~onfirn\cd to result in th< LZS[“CIc’Claltered amino .icid )I\ protein \equcncing (Table 3).
Do nuclear genes influence editing? Now that a fairly robust description of processing 4ites is a\-ailahlV for many genes in se\~er;ll organisms. resolution of the meclltini~mimportant for thib fornl of RXA pro-
cessing is cleat-ly the next step In the evrn more cQcnsively cditccl rnitt~chonclrtal \omes. guide
genes
ot
trypan
RNAs base pair to thtx rmedited Iran< ript and nicctiatt. tht correctIon of the prlmar\ trancnpt Like plant>. tr)panc,om& ai>o tIa\ c
qOMMENT TABLE 3. Verified amino acid changes in wheat AWP codon 7 28 75
DNA
Edited mRNA
TCA (Ser) CTC (Leu) CGA (Arg)
LJUA (Leu) UUC (Phe) UGA (Stop)
Amino
acid in protein Leu Phe None
“A. Araya, CNRS, Bordeaux.
~omplrx mitoc~hondri~~l gcnon~es. man) of the guide mttwstingly. RT.4q required for ‘CWV rtliting aw encoded by the minicircle component of the genome~. In the trypanosomes. editing proceeds in a strict S’+i’ fashion. and processing is thought to be c,omplete in the functional mRNAs because editing corrects many frameshifts. For cytochrome h. 102 of 106 cDNAs analysed were edited
on a manner consistent with this model. Adding an element of confusion. however, is the observation that about 4% of the COIZI transcripts displayed abn&mal editing (incorrect base changes and apparent deviation from the 3’-+5’ order)x. It is proposed that incorrect guide RNA molecules program the editing mistakes in C’O/fl tran4cripta. and one such aberrant guicie has been identified”. The tqpanosomc cmmple pro\,ides the model for future n-ork with higher plant editing and also allows some speculation about the current data. In particular. are the abundant partially edited trsnscripts in higher plants aberrantly edited transcripts? If so. is the underlying logic of editing obscured?
Future btmefit
studit ivith plants ma! ft-oni genetic analysis. cap-
t:lliTing on thr recrnr clisc~o\,erv that editing is inefficient and aberrant in a wheat male-sterile mutant (A. CytoAraya , CNRS. Bordeaux). plasmic male sterility (CMS) is a mitochondrial defect found in man) higher plants; the niitochondriall~ encoded lesion tllat results in pollen abortion can be corrected by dominant nuclear genes that restore fertility (Rf loci). In species such as maize with multiple types of CMS (caused by different defects in the mitochondrial genome), each CMS type is restored to fertility by different @loci. In a CM!? line of wheat, severe defects in atp’, editing were found. Not only is editing less conlplete than in normal organelles. but novel editing events also occur: (_;+A. A-G. and I-+Aio. At codon S7. a C+L> change results in a stop (.odon and truncated reading frame Normal editing is restored when the appropriate dominant nuclear restorer is introduced. Pollen sterility may be related to the massive faulty editing of the atp9 transcript (and perhaps other tnlnscripts as well). IWon-ing thiy argument. it is reasonable to propose that the
nuclear gene restorer encodes a guide RNA or another molecule required for normal atp9 editing. Cntil just a fern- years ago the suggestion that nuclear-encoded KNA molecules could traverse the mitochondrial membranes to function within the matrix wo~~lcl have seemed outlandish. Now. there arc several examples of required nuclear-encoded RNAs functioning in mitochondria. including the RN:I ~~omponent of the mouse RN.4 prt)cessing endorihonLlclcasel’l. and numerous tRNAs are imported from the cytoplasm into bean Rheat
mitochondriall. References I
2
6
7
8 9 10 II
Any Technical Tips? Technic4 Tips is :I place \vhere readers can exchange information ahout useful lab techniques. Technical Tips should he either new methods. or significant nen modifications or applications of existing methods. If you have developed a handy new method. why not share it with other 77G readers? Your article should be as brief as possible, but should give enough information to enable others to repeat the method. If an); part of the method involves published procedures. you can refer to the appropriate paper(s) rather than repeating those details. All Technical Tips are peer-reviewed. Please send three copies of your doul>le-spaced typescript. plus three copies of any figures (including at least one set of originals) to: Dr Alison Stemm. ‘IicmLs itl (Icwetk-s. tllse\kr Trends Journals. 68 Hills Road. Carnbridge CR2 lL4, UK
RNA editing in transcripts of higher plant mitochondria increases the similarity of the predicted proteins with products of yeast and mammalian genes. makes the predicted proteins nearly identical in different plant species. cam create start and stop codons in the open reading frames, and allows translation using universal codon rules. Editing is extensive, altering up to 13.50/o of the codons, and is concentrated in the coding regions. Many incompletely edited transcripts can be recovered as cDNAs from plant suggesting that mitochondria, protein polymorphism could exist. Editing is, however, more complete in spliced than in unspliced transcripts, and polysome-associated mRNAs are edited ‘to a much greater extent than the tOhI transcript pool.
Complexities of plant mitochondrial transcription The plant mitochondrial genome, ranging in complexity from about 200 to 2500 kb and containing ciris cular and linear molecules, almost terra incognita compared with the smaller mitochondrial genomes of fungi and animals’. Plant mitochondrial transcripts are also very complex. On northern blots, multiple transcripts (>lO per geue) are the rule rather than the exception in many species. In maize. the multiple tr:mscripty for XVeral genes originate from independent initiation e\renta at promoters from 100 bases to sc\cr:ti kh upstream of the coding region of a gene’. In addition to genonie and transcript complexity. codon usage in higher plant mitochondria seemed to present confusing esceptions to princ$ple\ established in the other kingdoms. The initial reports on RNA editing resolved two of the long-.standing difficulties in understanding J’lant mitochonclI-ial gene .structure. First. a proposed exception to uni\.ersat codon usage i\ unnt-ctL\\:ir!
At
positions of conserved tryptophan codons in mitochondrial genes of fungi. mammals and many plants, some exceptional plant genes had a CGG rather than a UGG codon: consequently. both IIGG and CGG were proposed to encode tryptophanl. This proposal created a second problem, however. as CGG was found at conserved Arg positions. suggesting that CGG could encode either Arg or Trp. With RN.4 editing. C+C con\ vrsion restores IJGG codons in the conserved tryptophan positions, making codon usage in higher plants consistent with the universal codej-5. Second, although plant mitochondrial genes of closely related species are nearly identical, radical amino acid substitutions were proposed from the deduced protein sequences. For example, the cytochrome oxidase subunit 2 (~0x2) genes of wheat and Inaize shon 98.90/o nucleotide identity. Hut the three differences in the 260 residue predicted amino acid sequence in\,olve radical replacements (maize--+wheat): Glu(GAA)+Ser(TCA), I’ro(CCA)--+Leu(CTT), and Cys(TGT)-+Arg(CGT). The apparent loss of the Cys was predicted to affect cytochrome oxidase function as this site is involved in binding a catalyticall)
essential copper ion 1, .4gain. RNA editing comes to the rescue, 21sthe Arg(CGU) codon is edited to Cys(UGU) in the wheat message.
Extensive editing in plant mitochondrial transcripts More news on RNA editing is now available from the Fourth International Workshop on Plant Mitochondria held on 23-2: September 1990 and hosted by Cornell L’niversity and the RO)XX Thompson Institute. By direct RYA sequencing or sequencing of uncloned cDNAs, transcripts representing about 50 kb of genes have been compared with genomic DNA. In other studies, multiple cloned cI)NAs have been sequenced to determine the distribution of edited sites within individual mRNAs. These studies hdVt! utilized primarily two monocots (wheat. maize) and t\vo dicots (Oenoth~ra, RWr7ia). The two largest data sets are sunlmarized in Tables 1 and 2. Important conclusiona from thr (Iat: availahte thus far include: ( 1) i\ll protein-coding transcripts examined to date are edited (2) The extent of editing varies Lvidely from two to 23 edits pc transcript. (3) The impact of editing can he extensive: in the r?nt/.3 transcript of wheat. 13.7X of the amino acid
TABLE1. Editing in wheat mitochondrial transcriptsa _ .~_______
Editing sites thneb
c+u
u+K
Altered amino acids Number
12
1
12
21 19
0 0
tpsl2
6
0
or-1 56
4
0
16 18 6 3
cox3 na& nad4
%
4.5 13.5 3.8 3.8 1.9
“The data (supplied by J-M. Grienenberger, CNRS, Strasbourg) refer to sites found to be edited in at least one cDNA; editing is not 100% efficient. “cox, cytochrome oxidase. subunit by number; nad, NADH oxidoreductase, subunit by number; rps. small ribosomdl subunit protein, number indicates approximate size in kDa; orj open reading frame.
fIG
T‘FIIRI,\RY
1991 \-()I. 7 \O. 1
mlOMMENT residues predicted from the DNA sequence are altered in the mRNA (Table 1). In the maize ~0~2 transcript, one codon receives two edits: CCA+UAA (KM. Mulligan. University of California, Irvine). (4) There is a strong bias (87% in \\hcat. El”,, in Oerzotheva) for editing that alters the amino acid specified by the codon. (5) In wheat all of the known editing sites are within the coding region. but in Oetzothera and maize. edited sites in Lhe 5’ and .?’ unlranslated region have been found. (6) Editing can also occur in structural RNA: the 26s rRNA of Oenothera has two edited sites. (7) Nearly all editing detected so far involves C--+(i changes (62 out of 63 in wheat: 118 out of 121 in Oenothera), but LJ+C edits have also been detected”. (8) Editing improv-es the degree of similarity at the level of predicted amino acid sequence for individual proteins among the higher plants as well as in comparisons between plants, fungi and animals.
TABLE2.
Gem? atp1
atpG up9 cob cox2 co3E3 nadl exon exon exon exon
A
B C D
exon E Maturase
nad3 rpsl4
orf
OrJB 1% rRNA 5S rRNA 26s rRNA
Codiig region length and transcript recognition features
For some plant mitochondrial genes, estimation of expected protein size has been problematical. Despite nucleotide similarity in the amino-terminal coding region. an ATG could be missing from the ‘obvious’ placct in t!~c DNA sequence as parison with
deduced
from
com-
other genes. At the carboxy terminus. predicted plant were proteins often virtual15 Identical through the region correspending to the cognate fungal and animal coding regions. but then the plant open reading frames diverged extensively and continued for variable distances, Such discrepancies in trdnSlational start and stop sites have now been resolved for several genes. For example, the ATG start codon for rzadl in wheat is created by RNA editing (ACG+AUG) (L. Honen, UniVet’Sity Of Ottawd). For the atp9 gene of wheat, CGG+ UGG editing creates a stop codon at apparent codon position 75. resulting in a 74 amino acid protein. Editing at RNA editing in Oemtbera position 75 also occurs in tl%UlSCtiptia the other plant species, making the predicted proNo. of Editingsites teins the same length. c-m u-6 altered AA@ Future studies may also indicate a role for editing 4 0 2 outside the coding region: 12 0 12 two possibilities would be 4 0 4 to increase the match to the 13 1 12 consensus sequence for 22 1 17 4 0 4 40s ribosome interaction or to alter intron splicing 4 0 4 kinetics. 1 0 0 9 0 6 1 11 4 16 2 5 7 0 0 1
0 0 0 o 0 0 0 0 0 1
I 8 4 13 2 4 5 NA NA NA
The data (supplied by W. Schuster and A. Brennicke, Institut fiir Genbiologische Forschung, Berlin) refer to sites found to be edited in at least one cDNA. bAbbreviations as for Table 1 plus cob, apocytochrome h; a proposed maturase gene is encoded within nadl. u
CAAS,amino acids.
Editing pattern Iri m(Jst tr~li~%XiptS, the
clislritW&)n ot‘ editing \itY\ I4 random within the c,oding region There is. however. a highly cIu.4tered pattern of edited \ites in wheat rrad4: this primary transcript has three axons of similar size. hut 1’ out of 19 editing sites are in the first exon. and just tn.0 are in cxon 3. 15 there an or&r to the editing process? From the first reports of RNA editing rn plant mltochondrial transcripts. it 5vas clear that although the majority of
potentially edited sites are edited, most mRNAs are incompietel) edited.+‘. By sequencing multiple cDNAs containing pre-edited sites. an order to RNA editing might be deduced. In plants such analyses have so far failed to elucidate any particular order to RNA editing. The available data suggest that at best some regions of transcripts are edited more rapidly or efficiently than others, but there is no apparent i’+_S’ or 3’%5’ order to the process.
Does editing produce fully edited mRNA? Among eight cDNAs of uad-3 from Oenothem, each transcript had at least two unedited sites (at random positions, out of a total of 16). The persistence of pre-edited transcripts and the observation that most edits alter a codon opens up the possibility that protein isoforms are encoded by the diverse transcripts from a single plant mitochondrial gene. There is experimental evidence suggesting, however, that translatable mRNAs are more completely edited than the total transcript pool. First. for transcripts undergoing both editing and intron splicing. editing is more complete in the spliced than unspliced molecules (C. Sutton and IM. Hanson, Cornell University). Second, polysomal RNA is almost completely edited (Mulligan). Thus, the functional mRNAs in plant mitochondria may each encode a single major protein form. Evaluation of the efficiency 01 editing and the uniformity of protein products will require analysis of mitochondrial proteins. Only for n.hcar tit]).9 ha\.c thrcr rclrting site5 ht,W ~~onfirn\cd to result in th< LZS[“CIc’Claltered amino .icid )I\ protein \equcncing (Table 3).
Do nuclear genes influence editing? Now that a fairly robust description of processing 4ites is a\-ailahlV for many genes in se\~er;ll organisms. resolution of the meclltini~mimportant for thib fornl of RXA pro-
cessing is cleat-ly the next step In the evrn more cQcnsively cditccl rnitt~chonclrtal \omes. guide
genes
ot
trypan
RNAs base pair to thtx rmedited Iran< ript and nicctiatt. tht correctIon of the prlmar\ trancnpt Like plant>. tr)panc,om& ai>o tIa\ c
qOMMENT TABLE 3. Verified amino acid changes in wheat AWP codon 7 28 75
DNA
Edited mRNA
TCA (Ser) CTC (Leu) CGA (Arg)
LJUA (Leu) UUC (Phe) UGA (Stop)
Amino
acid in protein Leu Phe None
“A. Araya, CNRS, Bordeaux.
~omplrx mitoc~hondri~~l gcnon~es. man) of the guide mttwstingly. RT.4q required for ‘CWV rtliting aw encoded by the minicircle component of the genome~. In the trypanosomes. editing proceeds in a strict S’+i’ fashion. and processing is thought to be c,omplete in the functional mRNAs because editing corrects many frameshifts. For cytochrome h. 102 of 106 cDNAs analysed were edited
on a manner consistent with this model. Adding an element of confusion. however, is the observation that about 4% of the COIZI transcripts displayed abn&mal editing (incorrect base changes and apparent deviation from the 3’-+5’ order)x. It is proposed that incorrect guide RNA molecules program the editing mistakes in C’O/fl tran4cripta. and one such aberrant guicie has been identified”. The tqpanosomc cmmple pro\,ides the model for future n-ork with higher plant editing and also allows some speculation about the current data. In particular. are the abundant partially edited trsnscripts in higher plants aberrantly edited transcripts? If so. is the underlying logic of editing obscured?
Future btmefit
studit ivith plants ma! ft-oni genetic analysis. cap-
t:lliTing on thr recrnr clisc~o\,erv that editing is inefficient and aberrant in a wheat male-sterile mutant (A. CytoAraya , CNRS. Bordeaux). plasmic male sterility (CMS) is a mitochondrial defect found in man) higher plants; the niitochondriall~ encoded lesion tllat results in pollen abortion can be corrected by dominant nuclear genes that restore fertility (Rf loci). In species such as maize with multiple types of CMS (caused by different defects in the mitochondrial genome), each CMS type is restored to fertility by different @loci. In a CM!? line of wheat, severe defects in atp’, editing were found. Not only is editing less conlplete than in normal organelles. but novel editing events also occur: (_;+A. A-G. and I-+Aio. At codon S7. a C+L> change results in a stop (.odon and truncated reading frame Normal editing is restored when the appropriate dominant nuclear restorer is introduced. Pollen sterility may be related to the massive faulty editing of the atp9 transcript (and perhaps other tnlnscripts as well). IWon-ing thiy argument. it is reasonable to propose that the
nuclear gene restorer encodes a guide RNA or another molecule required for normal atp9 editing. Cntil just a fern- years ago the suggestion that nuclear-encoded KNA molecules could traverse the mitochondrial membranes to function within the matrix wo~~lcl have seemed outlandish. Now. there arc several examples of required nuclear-encoded RNAs functioning in mitochondria. including the RN:I ~~omponent of the mouse RN.4 prt)cessing endorihonLlclcasel’l. and numerous tRNAs are imported from the cytoplasm into bean Rheat
mitochondriall. References I
2
6
7
8 9 10 II
Any Technical Tips? Technic4 Tips is :I place \vhere readers can exchange information ahout useful lab techniques. Technical Tips should he either new methods. or significant nen modifications or applications of existing methods. If you have developed a handy new method. why not share it with other 77G readers? Your article should be as brief as possible, but should give enough information to enable others to repeat the method. If an); part of the method involves published procedures. you can refer to the appropriate paper(s) rather than repeating those details. All Technical Tips are peer-reviewed. Please send three copies of your doul>le-spaced typescript. plus three copies of any figures (including at least one set of originals) to: Dr Alison Stemm. ‘IicmLs itl (Icwetk-s. tllse\kr Trends Journals. 68 Hills Road. Carnbridge CR2 lL4, UK
