[]~EVIEWS 19 Mackman, N., Nicaud, J.M., Gray, L. and Holland, I.P. (1986) Curr. Top. Microbiol. ImmunoI. 125, 159-181 20 Strathdee, C.A. and Lo, R.Y.C. (1989)J. Bacteriol. 171,
Acad. Sci. USA 87, 4776-4780 32 Higgins, C.F. (1990) Res. Microbiol. 41,353-360 33 Endicott, J. and Ling, V. (1989) Annu. Rev. Biochem. 58,
137-171
916-928 21 Glaser, P. et al. (1988) EMBOJ. 7, 3997-4004 22 Fath, M.J., Skvirsky, R.C. and Kolter, R. J. Bacteriol. (in 23 24 25 26 27 28 29 30 31
34 Kuchler, K., Sterne, E.S. and Thorner, J. (1989) EMBOJ.
8, 3973-3984
press) Economou, A., Hamilton, W.D.O., Johnston, A.W.B. and Downie, J.A. (1990) FA4BOJ. 9, 349-354 L6toffe, S., Delepelaire, P. and Wandersman, C. (1990) ~ B O J . 9, 1375-1382 Letoffe, S., Delepelaire, P. and Wandersman, C. (1991) J. Bacteriol. 173, 2160-2166 Guzzo, J. et al. (1991) Mol. Microbiol. 5, 447-453 Delepelaire, P. and Wandersman, C. (1990) J. Biol. Chem. 265, 17118-17125 Stanley, P., Koronakis, V. and Hughes, C. (1991) Mol. Microbiol. 5, 2391-2405 Delepelaire, P. and Wandersman, C. (1991) Mol. Microbiol. 5, 2427-2435 Guzzo, J. et al. J. Bacteriol. (in press) Wandersman, C. and Delepelaire, P. (1990) Proc. Natl
35 Kelly, A. et al. (1992) Nature 355, 641-644 36 Kenny, B., Haig, R. and Holland, I.B. (1991) Mol. Microbiol. 10, 2557-2568 37 Letoffe, S. and Wandersman, C. J. Bacteriol. (in press) 38 Michiels, T. et al. (1991) J. Bacteriol. 173, 4994-5009 39 Schiebel, E., Schwarz, H. and Braun, V. (1989)J. Biol. Chem. 264, 16311-16320 40 Kuehn, M.J., Normark, S. and Hultgren, S.J. (1991) Proc. Natl Acad. Sci. USA 88, 10586-10590 41 Vogler, A.P., Homma, M., Irikura, V.M. and Macnab, R.M. (1991) J. Bacteriol. 173, 3564-3572
C, WANDERSMANIS IN THEUNITI~DEGI~N~TIQUEMOLI~CULAIRE (CNRS UA 1149), INSTITUTPASTEUR, 25 RUEDU DR ROUX, 75724 PARISCEDEX15, FRANCE.
Regenerating good sense: I n their molecular biology, higher plant mitochondria have always stood a little apart from their mammalian, fungal, trypanosome or even algal equivalents. In particular, their complex genome organization and large coding potential prompted speculation about what genes were actually present and expressed in these organelles. The molecular biology of plant mitochondria has gained added interest with the discovery of mRNA editing in these organelles. Plant mitochondria edit almost all of their mRNAs 1-3, considerably more frequently than chloroplasts do, and in a manner rather different from RNA editing in other organisms such as trypanosome mitochondria and the mammalian cytoplasm. RNA editing in chloroplasts appears to be similar to the editing in plant mitochondria and may turn out to employ the same nuclear-encoded factors 4,5. The assembly of mature transcripts from several mRNA molecules by trans splicing also occurs in both organelles, but is far more frequent in plant mitochondria. Such trans-splicing reactions may compensate for the frequent rearrangements that occur in plant mitochondrial genomes. RNA editing RNA editing in plant mitochondria was indicated by discrepancies between genomic and cDNA sequences 1-3. Specifically, for some genomic nucleotides that were predicted to be transcribed as cytidines, the cDNA sequence indicated a uridine in the mRNA. The observed differences between cDNA and genomic sequences are almost exclusively C-to-T alterations, indicating C-to-U changes in the RNA, but U-to-C changes have also been reported 6,7. The C-to-U alterations have been found in almost all of the identified open l:eading frames in mitochondria of all higher plant species investigated to date. The exception is the T-urf13 gene, which is created by recombination in the male-sterile CMS-T cytoplasm of
RNAediting and trans splicing in plant mitochondria BERND WISSINGER, AXEL BRENNICKEAND WOLFGANG SCHUSTER The protein products of plant mitocbondrial genes cannot be predicted accurately from genomic sequences, since RNA editing modifies almost all mRNA sequences posttranscriptionally. Furthermore, RNA editing alters leader, trailer and intron sequences, and may be required for processing of these sequences. For several plant mitochondrial transcripts, processing includes trans spllcin~ which connects exons scattered throughout the genome. The mature transcripts are assembled via split group II intron sequences.
maize 8. The species for which RNA editing has been demonstrated include both monocots and dicots, for example Arabidopsis, carrot, maize, Oenothera, pea, Petunia and wheat. In fact, RNA editing in plant mitochondria appears to pre-date the divergence of the Pteridophyta (fernqike plants) and Spermatophyta (seed plants) and may thus be an ancient trait (R. Hiesel, B. Combettes and A. Brennicke, unpublished). The genomic sequence of the bryophyte Marchantia 9 suggests that RNA editing is not required in this moss. The rRNAs in higher plant mitochondria have been partially analysed by direct RNA sequencing in wheat 1° and by cDNA analysis in Oenothera n. Neither investigation revealed any definite indication of editing. One cDNA clone of the 26S rRNA in Oenothera mitochondria was found to differ from the genomic sequence at two positions11: one C to U and one reverse event located close together in a loop structure. Since these alterations were found in only one of several clones, their significance is unclear.
TIG SEPTEMBER 1 9 9 2 VOL. 8 NO. 9 ©1992 Elsevier Science Publishers Ltd (UK)
tm
VlEWS It is not known whether tRNAs are edited. Comparison of tRNA genes in different species shows C-to-U transitions at some positions that may be compensated for by RNA editing. Unfortunately, since direct sequencing of several tRNAs was done in species different from those for which genomic sequences are available, no clear deductions about editing in tRNAs can be made. All of the protein-coding mRNAs investigated in higher plant mitochondria are with the exception of T-urfl3- edited to some extent. The degree of editing, however, differs considerably for different genes and in different species. The most complete set of data is currently available for Oenothera mitochondria, where more than 20 genes have been investigated for RNA editing (Fig. 1). The nad3 mRNA is one of the most extensively edited transcripts, with 4.5% of all nucleotides altered in Oenothera 13 and 13% of the amino acids altered in wheat mitochondria 14. The least edited open reading frame is atpA in Oenothera, where only 0.25% of the nucleotides are edited n.
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The C-to-U alterations made by RNA editing will obviously affect only codons containing C residues. Most frequently FIGn altered are first and second codon positions RNA editing sites in Oenotheramitochondria are clustered in open reading that change the identity of the encoded frames. Each experimentally determined editing site is indicated by an arrow. amino acid. The functional importance of Closed boxes represent the open reading frames of individual genes and exons analysed for editing, dashed open boxes parts of genes not analysed. the less frequent silent changes at the third Solid lines represent noncoding sequences analysed for RNA editing. Exons codon position is as yet unclear. Of obvious importance are alterations creating AUG are marked in alphabetic order. Abbreviations: atpA, atp6, atp9, ATPase initiation codons, as in the had1 mRNA of subunits; cob, cytochrome b; coxI, coxlI, coxIII, cytochrome c oxidase subunits; mat-r, maturase-related reading frame; nadl, nad3, nad5, NADH wheaO 5 and in chloroplasts 4,5, and editing dehydrogenase subunits; orf, orfB, open reading frame; rps3, rpsl3, rpsl4, events resulting in termination codons. rpsl9, rpl5, ribosomal proteins of the small or large subunit; 5S rRNA, 18S Analysis of the atp9 gene product in wheat rRNA, 26S rRNA, ribosomal RNAs. mitochondria revealed that the UGA termination codon created by RNA editing resulted in a correspondingly shortened polypeptide ]6. in Oenothera, for example, is partially edited in several RNA editing has resolved the long-standing debate positions. The deduced translation products of both partially and fully edited mRNAs show similarity with about whether plant mitochondria have a nonstandard the corresponding proteins of several other species 9, genetic code. From genomic sequence comparisons, suggesting that in theory proteins synthesized from both CGG and the normal triplet UGG had been suspected to code for tryptophan 17As. Investigation of RNA both types of mRNAs could be functional. editing has shown that nearly all CGG codons at posThe only plant mitochondrially encoded polypeptides for which sequence data are available are the itions where tryptophan is evolutionarily conserved are altered to UGG. Thus there is no longer any need to amino-terminal portion of the coxIIgene product from sweet potato ]9, and the atp9 gene product from postulate a divergent genetic code in plant mitochonwheat16, 20. The former gives no indication of RNA editdria. ing, while the latter confirms all editing sites determined by cDNA analysis. There is no indication of hetProtein families by differential editing? erogeneity in the amino acid sequence of the atp9 For several plant mitochondrial genes, comparisons of independent cDNA clones show that transcripts of gene product, in agreement with the observation that the same gene may be edited to different degrees. If all editing sites in the wheat atp9 transcripts appear to be completely edited in the mRNA pool. In the case of all these mRNAs were equally accessible for transgenes for which a high percentage of partially edited lation, slightly different polypeptides could be synmRNAs has been observed, protein sequence data are thesized. The transcript population of the nad3 gene TIG SEPTEMBER 1 9 9 2 VOL. 8 NO. 9
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[]~EVIEWS NUCLEOTIDE REPLACEMENT
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FIG[] The biochemical reactions possible for RNA editing in plant mitochondria. (A) Deamination of a specific cytidine in the RNA chain catalysed by a cytidine deaminase activity. (B) Removal of the cytosine base by an RNA glycosylase activity without cleavage of the sugar-phosphate backbone and the subsequent insertion of an uracil base. (C) Nucleotide replacement by excision of a C and insertion of a U. The parts of the molecules affected by the potential reactions are circled.
OH OH 3'
required to resolve the question of w h e t h e r variant p o l y p e p t i d e s can be synthesized from a single gene by differential RNA editing.
How does RNA editing work in plant mitochondria? The biochemistry of RNA editing in plant mitochondria is as yet unclear. Three different reactions could theoretically be responsible for the C-to-U transitions: deamination, base substitution and nucleotide excision and insertion (Fig. 2). DNA deamination is a spontaneous reaction and a c o m p l e x repair system has .evolved to eliminate the errors introduced by it. Deamination of a C to U alters the sequence of the apolipoprotein B mRNA in the cytoplasm of some mammalian tissues, a reaction that
appears to be mediated by a single p o l y p e p t i d e 2122. Furthermore, cytidine m o n o n u c l e o t i d e deaminase activities have b e e n found in higher plants, but are not localized to any c o m p a r t m e n t and have not b e e n tested for their activity on polynucleotides. The rare reverse events might require a different enzymatic catalysis in the form of a CTP synthase activity. The close proximity of C-to-U and U-to-C editing events in a 26S rRNA cDNA clone in Oenothera could suggest a transamination mechanism that transferred the amino group from the C to the U residue in the same rRNA molecule. However, such a reaction w o u l d require n e w enzymatic activities and remains speculative. The second possibility is that editing could p r o c e e d via excision of the base without interrupting the
TIG SEPTEMBER1992 VOL. 8 NO. 9
m
[]~EVlEWS
sugar-phosphate backbone. Such RNA glycosylation reactions have been described in tRNAs and it is plausible that they may also alter mRNA sequences in plant mitochondria. The third possible pathway involves a complete cut of the nucleotide chain, excision of the cytidine, addition of a uridine and subsequent religation of the interrupted molecule. Trypanosome mitochondria add and delete uridines in their mRNAs by such a mechanism, which involves transesterifications in the insertion and religation steps 23,24. However, numerous intermediates have been identified in trypanosome mitc,chondria in which the number of uridines deviates from the final sequence. No clear evidence for such aberrations has yet been observed in any of the completely or incompletely edited plant mitochondrial transcripts, even though large numbers of cDNA clones and sequences amplified by the polymerase chain reaction (PCR) have been investigated.
Which nucleotide is edited? The specificity of RNA editing in plant mitoch6hdria is probably determined independently from the biochemical activity. The many different editing sites without any overt similarity in the surrounding nucleotide sequences seem to require a complex mechanism for determining specificity. The editing event(s) in the apolipoprotein B mRNA are recognized solely in their sequence context by a protein factor22. In contrast, no common motifs can be discerned in the vicinity of RNA editing sites in plant mitochondria apart from a loose bias in nucleotide distribution, with guanosines found only rarely as the nucleotide preceding an edited cytidine25, 26. The species-specific editing events observed in identical homologous genes rule out the primary sequences immediately surrounding editing sites as the sole determinants of an editing site. Although some short sequence similarities have been identified between selected edited regions, no consensus sequence could be deduced that covered all known editing sites 7. RNA editing in plant mitochondria does not proceed in a single direction along the mRNA molecule, but rather appears to select the nucleotide that is to be edited by differential affinity to individual sites. Antisense RNA molecules unique for each site could provide the specificity of plant mitochondrial editing, analogous to the guide RNA model for trypanosome mitochondria 23,24. Guide RNA molecules for editing in plant mitochondria have not yet been identified although they have been looked for extensively. If they exist, their sequence specificity would not require more than nine or ten nucleotides for base pairing to allow unambiguous definition of a given editing site - and such short sequence similarities may be difficult to identify.
Are some RNA editing events mistakes? The identification of several RNA editing events that have no apparent advantage may provide some further clues to the specificity determinants of RNA editing in plant mitochondria. A pseudogene of rps19 in Oenothera mitochondria 27 is edited at positions differing from the editing sites of the functional open reading frame in Petunia mitochondria 2s. Only one
site is conserved, and this is a silent nucleotide position. Most of the other editing sites in the functional gene in Petunia improve the evolutionary conservation of the protein, while the editing events in the rpsl9 pseudogene in Oenothera lessen the degree of similarity but are nevertheless specific to certain cytidine residues. This finding suggests that the editing specificity can shift from one nucleotide position to other(s) if functional pressure is released. Apparent 'mistakes' in editing specificity have also been observed in essential coding regions in spite of a functional requirement for these proteins. Termination codons have been found after editing in the first few triplets of coxI, atp6and rps3 mRNAs in Oenothera mitochondria, presumably precluding synthesis of the encoded polypeptides 27. These 'mistakes' are as (in)frequent as some of the silent editing events, which might likewise originate from such a loose specificity, but may be tolerated because they do not affect the translation product. Introduction of translational stops by editing may on the other hand have some regulatory function in modulating the translatable RNA pool.
Editing in plant mitochondria is post-transcriptional The first indications that RNA editing in plant mitochondria was post-transcriptional came from analysis of individual cDNA clones from several protein-coding genes that were found to be differentially edited 13. Such partially edited molecules may represent editing intermediates that are further edited to the mature mRNA molecules. In maize and Petunia mitochondria x9,3°, spliced coxlI transcripts are more extensively edited than unspliced molecules. The large transcripts of nad3 in wheat - presumed to be the precursor molecules - are less extensively edited than a smaller transcript of this gene that may be a processing produc0 4. These observations suggest that RNA editing is a true posttranscriptional process. Cis
and trans splicing in plant mitochondria
In addition to RNA editing, complex splicing processes are required to create meaningful open reading frames for some plant mitochondrial polypeptides. The presence of introns was documented when the first plant mitochondrial gene was identified and sequenced 17. All higher plant mitochondrial introns that have been described show typical features of group II introns and can be folded into the consensus model structure. Group I introns have not yet been detected in these organelles. To date, about a dozen cis-splicing introns have been found in plant mitochondria and more are likely to turn up as new genes are identified (Table 1). The identification of genes is made particularly complicated by transsplicing events such as those recently found in wheat, maize, Petunia, Oenothera and Arabidopsis mitochondria 15,31,32 (Table 1). These trans-splicing events occur exclusively in the transcripts encoding subunits of the NADH dehydrogenase complex (Table 1). The complete open reading frame of the nadl gene is encoded by five exons, connected by two cis- and two trans-splicing events
TIG SEPTEMBER 1 9 9 2 VOL. 8 NO. 9
[]~EVIEWS
The plant mitochondrial trans-splicing events appear mostly to pre-date the divergence of at least monocots and dicots. Some trans-splicing events, however, must be of recent origin, since Tra~s Intron the wheat, Petunia and Oenothera open reading inttons ~ editing Ref. frames differ in their organization and in the ~ I (wheat/Petun/a) 1 3 ? 15.32 number of trans-splicing events (Fig. 4). The ~o bidopsts) 2 2 ÷/? 31a cis-spliced intron between exons d and e of nadl requires an additional trans-splicing event (~otbera) 3 1 + 35 in wheat and Petunia. These trans-splicing introns possibly result from the frequent recombinad4 (wheat/Arabidopsis) 3 ? 40~ nation within these plant mitochondrial genomes rearrangements that have disrupted the once had5 ( ~ idopsis) 2 2 ÷ 33 c/s-splicing introns without deleterious consequences as long as the open reading frame can t i o n s : - , no intron of the respective ~ present; +, intron e d i ~ g identified: ?, intron sequences not investigated. be assembled in trans. Gene ature is as m Fig. 1. The specificity of trans splicing is possibly ~W, Schuster and A. Brennicke, unpublished, associated with a slightly longer base-pairing stem of the interrupted domain IV. These pairings connect the two mRNA molecules to in Oenothera, but one cis- and three trans-splicing reconstitute the former cis-splicing intron that was events in wheat and Petunia. The nad5 transcripts in broken by genomic recombination 15,31. Experimental Oenothera, Arabidops/s, wheat and maize require two evidence is still required, however, to show that these cis- and two trans-splicing events to integrate an exon sequences are involved in the specificity of trans splicof only 22 nucleotides33,34. One trans-splicing reaction ing. It has been suggested that such split group II and three cis-splicing events are needed to connect the introns may represent the first steps in the evolution of open reading frame of the nad2 gene in Oenothera nuclear intron splicing, where most of the activities and Arabidops/s35. have been dissociated from the actual intron and n o w The trans-splicing reactions in plant mitochondria function in trans. appear to be associated with the presence of intron sequences that can be aligned by base pairing to form RNA editing in i n t r o n s an interrupted typical group II intron structure. The Intron splicing and RNA editing appear to be insecondary structure models place the discontinuity in dependent yet interdependent post-transcriptional prothe mitochondrial trans-splicing introns in domain IV cesses in plant mitochondria. Several intron sequences (Fig. 3). This location is different from the transappear to require RNA editing in order to fold into the splicing intron model of rps12 in higher plant chlorocorrect secondary structure, which is presumably plasts36, where the intron sequence is interrupted in essential for function. domain III, but is similar to the psaA transcripts in Two RNA editing events have been observed31 in Chlamydomonas chloroplasts37, where an intron is the 3' region of the trans-splicing intron a/b in the fragmented in domains I and IV. nadl mRNAs of Oenothera that appear to be essential for folding of domain VI. Other editing events in the stem regions of domain VI were found in a cis-splicing intron of nad5 domain III mRNA and in the trans-splieing nadl intron c/d; both of these events are also needed to maintain the conserved base-paired configuration31,33. The single trans-spliced intron sequence of the nad2 gene is edited once in domain I and once in the downstream stem part of domain IV (Ref. 35). The edited nucleotide in domain I appears to be indispensable in defining this stem and therefore to be essential for intron folding and excision. Both editing sites observed in the nad2 intron sequences are edited as frequently as non-silent editing sites in open v, reading frames, further supporting their functional importance. These intron editing events have so far only been determined in intron sequences of FIG[] unspliced precursor mRNAs. The complete Secondary structure models of the nucleotide sequences flanking trans-splicing exons in plant mitochondria suggest the involvement of group set of RNA editing sites required for a functional intron structure, however, can only be II intron sequences. These models localize the disruption in the structure model in domain IV, one of the variable regions of such introns. determined in the excised intron sequence.
m.,o,v
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TIG SEPTEMBER 1 9 9 2 VOL. 8 NO. 9
~2(
r~EV. IEWS
Whyeditingand splicing? In the conventional dogma pre-mRNA 2 'DNA makes RNA makes protein', there is no obvious place either for RNA editing "~. "~..~" ,~" or for introns. The arguments mature mRNA • I = Iblcl°l I invoked to rationalize the ..,¢ . f.,¢'~. '~. existence of introns (evol.." ...::::-'" ".. ".. utionary advantage, selfish ~-..-:2-';"" . """ pre-mRNA1 - - t a propagation, control of gene expression) must also be inpre-mRNA 3, voked for RNA editing. At mat.-r. present we can barely even speculate on any 'selfish' Petunia attribute of RNA editing, unless it perhaps confers an pre-mRNA 3 I-advantage on the organelle itself, protecting its rudipre-mRNA 4 mentary genome from slipping into the nucleus. Against this wheat suggestion, edited versions of lost or truncated mitopre-mRNA 3 [~-chondrial genes have been found to be encoded by nupre-mRNA 4 I mat.-r. clear genes, exemplified by the coxII gene in pea38 and the rpsl2 gene in Oenothera pre-mRNA 1 (L. Grohmann, A. Brennicke and W. Schuster, unpublished). , Less far-fetched may be a connection with regulatory mature mRNA --] a I mechanisms that could act b °41 ...... .... "% through RNA editing to alter the pool of translatable pre-mRNA 2 mRNAs. Several RNA editing events in plant mitochondria pre-mRNA 3, suggest just such a connection. I ° i The examples of RNA editing in intron sequences discussed FIG[F] in the previous section are the Top: Exons of the nadl genes in Oenotheraz:, Arabidopsis (W. Schuster, unpublished), most obvious candidates, since Petunia 32 and wheat 15 require several trans~plicing events for assembly of the open reading frame. The two trans~.plicing reactions between exons a/b and c/d are conserved their requirement for splicing in all species, whereas the Oenotbera and Arabidopsis cis-splicing intron between exons die could be used to regulate is interrupted in wheat and Petunia in different positions, resulting in an additional the levels of spliced, mature trans-splicing event in these plants. In the latter two species the mature nadl mRNAis transcripts. Another example thus assembled from four pre-mRNA molecules. Such trans-splicing events are probably of a potential regulatory funcresults of the recombination events that frequently rearrange plant mitochondrial genomes. tion for an editing event in Bottom: Assembly of the mature mRNAfor the had5 gene in Oenotbera and Arabidopsis a noncoding sequence could from three different pre-mRNAmolecules. This gene is organized similarly in wheat and be provision of an improved maize. Abbreviations are as in Fig. 1. ribosome-binding site upstream of rpsl4 (Ref. 39). The first editing event first triplets of coxI and rps3 open reading frames in the n a d l open reading frames creates an AUG could be examples of such regulation. codon, the only potential start codon in wheat 15. Presumably the unedited transcript cannot be transAcknowledgements lated, and RNA editing at this site would thus be the We thank Stefan Binder, Lutz Grohmann, Rudolf Hiesel, limiting step in production of a translatable mRNA Volker Knoop and Anita Marchfelder for discussions and unpublished data. Work in our laboratory was supported by molecule. grants from the Deutsche Forschungsgemeinschafl and the Thus RNA editing could, at least in some instances, Bundesministerium for Forschung. provide a rapid transition from a pool of steady-state unedited and untranslatable mRNA to mature mRNAs, References without any de novo synthesis of transcripts. RNA edit1 Covello, P.S. and Gray, M.W. (1989) Nature 341, 662466 ing may also be able to regulate the reverse step, 2 Gualberto, J.M. et al. (1989) Nature 341,660-662 making mRNAs untranslatable and condemning them 3 Hiesel, R., Wissinger, B., Schuster, W. anti Brennicke, A. to degradation. The termination codons found in the (1989) Science 246, 1632-1634 L
i
TIG SEPTEMBER1992 VOL.8 NO. 9 ~27
4 Hoch, B. etal. (1991) Nature353, 178-180 5 Kudla, J. etal. (1992)EMBO.L 11, 1099-1103 6 Schuster, W., Hiesel, R., Wissinger, B. and Brennicke, A. (1990) Mol. Cell. Biol. 10, 2428-2431 7 Gualberto, J.M., Well, J-H. and Grienenberger, J-M. (1990) Nucleic Acids Res. 18, 3771-3776 8 Ward, G.C. and Levings, C.S., III (1991) PlantMol. Biol. 17, 1083-1088 9 0 d a , K. etal. (1992)J. Mol. Biol. 223, 1-7 10 Spencer, D.F., Schnare, M.N. and Gray, M.W. (1984) Proc. Natl Acad. Sci. USA 81,493-497 11 Schuster, W. et al. (1991) Curr. Genet. 20, 397-404 12 Welt, J-H. (1988) PlantMol. Biol. Rep. 6, 30-31 13 Schuster, W., Wissinger, B., Unseld, M. and Brennicke, A. (1990) EMBOJ. 9, 263-269 14 Gualberto, J.M., Bonnard, G., Lamattina, L. and Grienenberger, J-M. (1991) Plant Cell 3, 1109-1120 15 Chapdelaine, Y. and Bonen, L (1991) Cell 65, 465-472 16 Begu, D. el al. (1990) Plant Cell 2, 1283-1290 17 Fox, T.D. and Leaver, C.J. (1981) Cell26, 315-323 18 Hiesel, R. and Brennicke, A. (1983) EMBOJ 2, 2173-2178 I 9 Maeshima, M., Nakagawa, T. and Asahi, T. (1989) Plant Cell Physiol. 30, 1187-1188 20 Graves, P-V. et al. (1990) J. Mol. Biol. 214, 1-6 21 Powell, L.M. etal. (1987) Cell50, 831~840 22 Greeve, J., Navaratnam, N. and Scott, J. (1991) Nucleic Acids Res. 13, 3569-3576 23 Blum, B., Bakalara, N. and Simpson, M. (1990) Cell 57, 355-366 24 Blum, B., Sturm, N.R., Simpson, A.M. and Simpson, L. (1991) Cell65, 543-550
25 Covello, P.S. and Gray, M.W. (1990) Nucleic Acids Res. 18, 5189-5196 2 6 Schuster. W. et al. (1991) Physiol. Plant. 81,437~i45 2 7 Schuster, W. and Brennicke, A. (1991) Nucleic Acids Res. 19, 6923-6928 28 Conklin, P. and Hanson, M.R. (1991) Nucleic Acids Res. 19, 2701-2705 29 Sutton, C.A., Conklin, P., Pruitt, K.D. and Hanson, M.R. (1991) Mol. Cell. Biol. 11, 4274-4277 3 0 Yang, A.J. and Mulligan, M. (1991) Mol. Cell. Biol. 11, 4278~281 31 Wissinger, B., Schuster, W. and Brennicke, A. (1991) Cell 65, 473-482 32 Conklin, P.L., Wilson, R.K. and Hanson, M.R. (1991) Genes Dev. 5, 1407-1415 3 3 Knoop, V., Schuster, W., Wissinger, B. and Brennicke, A. (1991) ~ B O J . 10, 3483-3493 35 Binder, S., Marchfelder, A., Brennicke, A. and Wissinger, B. (1992) J. Biol. Chem. 267, 7615-7623 3 6 Fukuzawa, H. et al. (1986) FEBSLett. 198, 11-15 3 7 Goldschmidt-Clermont, M. et al. (1991) Cell65, 135-143 3 8 Nugent, J.M. and Palmer, J.D. (1991) Cell66, 473-481 39 Schuster, W., Unseld, M., Wissinger, B. and Brennicke, A. (1990) Nucleic Acids Res. 18, 229-233 4 0 Lamattina, L. and Grienenberger, J-M. (1991) Nucleic Acids Res. 19, 3275-3282 B. WISSINGER, A. BRENNICKEAND W. SCHUSTER ARE IN THE INSTITUT Ff2R GENBIOLOGISCHEFORSCHUNG, IHNESTRASSE 63, D-IO00 BERLIN33, FRG.
] • ! g A .
Chickens in search of a market
Genetics and Evolution of the Domestic Fowl by Lewis Stevens Cambridge University Press, 1991. £50, $89.95 (xiii + 306 pages) ISBN 0 521 40317 0 Most genetics textbooks focus on the subject itself, drawing examples from whichever taxonomic group can best exemplify the point under consideration. Occasionally, however, a book appears that is restricted to a particular species one that attempts to discuss the current state of the art (sorry, science) for that organism. This is just such an attempt, and is a splendid little book that is the first devoted to the domestic fowl for many years. Because of its very nature, Genetics and Evolution of the Domestic Fowl covers a wide range of topics. The opening chapter discusses the evolutionary history of the species, and describes the contribution of genetics to its elucidation. The results support the general consensus from anatomy and behaviour that the closest relative of the domestic fowl is probably the Red Jungle Fowl (Gallus gallus), but the possibility of a polyphyletic origirr cannot be ruled out. There then follow chapters on genetic principles, cell biology, mendelian inheritance, sex
linkage and gene mapping to set the scene for the more detailed sections on anatomical genetics - feathering, plumage variation, nerves, musculature, etc. A very good chapter on quantitative genetics shows how the poultry industry has applied selection and environmental management to improve the product out of all recognition in the past 20 years. The book then moves on to the really technical stuff- protein evolution, immunogenetics, gene cloning and transfer, and a final appendix on oncogenes. As I said, an excellent little book, but who will buy it? If they have any sense, commercial breeders will hire a professional geneticist to help them through the morass of responses to selection, immunization procedures, and so on. But there is also a whole subculture out there of folk who enjoy keeping and breeding chickens for pleasure and show. Might they be persuaded to buy? I doubt it. I have some knowledge of a parallel group who keep pedigree dogs, and its T1G SEPTEMBER 1 9 9 2 VOL. 8 NO. 9
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aficionados would find a book on dogs pitched at an equivalent level very hea W going. The sections on basic inheritance and linkage would be useful, but a little more on the differentiation of breeds would probably have been welcome to this rather specialist, if nontechnical, market. The chapter on quantitative genetics would be an excellent basis for a short course in animal improvement, but it is perhaps expecting a great deal of a student to buy the book for a single chapter. The later sections are also excellent reviews of those aspects of molecular genetics relevant to birds in general, and fowls in particular. I think, though, that the background theory is equally well presented in more general texts. So I wonder where the market lies? Despite these reservations, I really enjoyed this book, and will refer to it frequently. My interest in birds has not extended to their immunology or oncogenetics, and it fills that gap to perfection. I will also use the chapter on quantitative genetics in my teaching, so it will not gather dust on the shelf as so many other books do.
David T. Parkin Department of Genetics, School of Medicine, Queen's Medical Centre, Nottingham, UKNG72UH.
Acad. Sci. USA 87, 4776-4780 32 Higgins, C.F. (1990) Res. Microbiol. 41,353-360 33 Endicott, J. and Ling, V. (1989) Annu. Rev. Biochem. 58,
137-171
916-928 21 Glaser, P. et al. (1988) EMBOJ. 7, 3997-4004 22 Fath, M.J., Skvirsky, R.C. and Kolter, R. J. Bacteriol. (in 23 24 25 26 27 28 29 30 31
34 Kuchler, K., Sterne, E.S. and Thorner, J. (1989) EMBOJ.
8, 3973-3984
press) Economou, A., Hamilton, W.D.O., Johnston, A.W.B. and Downie, J.A. (1990) FA4BOJ. 9, 349-354 L6toffe, S., Delepelaire, P. and Wandersman, C. (1990) ~ B O J . 9, 1375-1382 Letoffe, S., Delepelaire, P. and Wandersman, C. (1991) J. Bacteriol. 173, 2160-2166 Guzzo, J. et al. (1991) Mol. Microbiol. 5, 447-453 Delepelaire, P. and Wandersman, C. (1990) J. Biol. Chem. 265, 17118-17125 Stanley, P., Koronakis, V. and Hughes, C. (1991) Mol. Microbiol. 5, 2391-2405 Delepelaire, P. and Wandersman, C. (1991) Mol. Microbiol. 5, 2427-2435 Guzzo, J. et al. J. Bacteriol. (in press) Wandersman, C. and Delepelaire, P. (1990) Proc. Natl
35 Kelly, A. et al. (1992) Nature 355, 641-644 36 Kenny, B., Haig, R. and Holland, I.B. (1991) Mol. Microbiol. 10, 2557-2568 37 Letoffe, S. and Wandersman, C. J. Bacteriol. (in press) 38 Michiels, T. et al. (1991) J. Bacteriol. 173, 4994-5009 39 Schiebel, E., Schwarz, H. and Braun, V. (1989)J. Biol. Chem. 264, 16311-16320 40 Kuehn, M.J., Normark, S. and Hultgren, S.J. (1991) Proc. Natl Acad. Sci. USA 88, 10586-10590 41 Vogler, A.P., Homma, M., Irikura, V.M. and Macnab, R.M. (1991) J. Bacteriol. 173, 3564-3572
C, WANDERSMANIS IN THEUNITI~DEGI~N~TIQUEMOLI~CULAIRE (CNRS UA 1149), INSTITUTPASTEUR, 25 RUEDU DR ROUX, 75724 PARISCEDEX15, FRANCE.
Regenerating good sense: I n their molecular biology, higher plant mitochondria have always stood a little apart from their mammalian, fungal, trypanosome or even algal equivalents. In particular, their complex genome organization and large coding potential prompted speculation about what genes were actually present and expressed in these organelles. The molecular biology of plant mitochondria has gained added interest with the discovery of mRNA editing in these organelles. Plant mitochondria edit almost all of their mRNAs 1-3, considerably more frequently than chloroplasts do, and in a manner rather different from RNA editing in other organisms such as trypanosome mitochondria and the mammalian cytoplasm. RNA editing in chloroplasts appears to be similar to the editing in plant mitochondria and may turn out to employ the same nuclear-encoded factors 4,5. The assembly of mature transcripts from several mRNA molecules by trans splicing also occurs in both organelles, but is far more frequent in plant mitochondria. Such trans-splicing reactions may compensate for the frequent rearrangements that occur in plant mitochondrial genomes. RNA editing RNA editing in plant mitochondria was indicated by discrepancies between genomic and cDNA sequences 1-3. Specifically, for some genomic nucleotides that were predicted to be transcribed as cytidines, the cDNA sequence indicated a uridine in the mRNA. The observed differences between cDNA and genomic sequences are almost exclusively C-to-T alterations, indicating C-to-U changes in the RNA, but U-to-C changes have also been reported 6,7. The C-to-U alterations have been found in almost all of the identified open l:eading frames in mitochondria of all higher plant species investigated to date. The exception is the T-urf13 gene, which is created by recombination in the male-sterile CMS-T cytoplasm of
RNAediting and trans splicing in plant mitochondria BERND WISSINGER, AXEL BRENNICKEAND WOLFGANG SCHUSTER The protein products of plant mitocbondrial genes cannot be predicted accurately from genomic sequences, since RNA editing modifies almost all mRNA sequences posttranscriptionally. Furthermore, RNA editing alters leader, trailer and intron sequences, and may be required for processing of these sequences. For several plant mitochondrial transcripts, processing includes trans spllcin~ which connects exons scattered throughout the genome. The mature transcripts are assembled via split group II intron sequences.
maize 8. The species for which RNA editing has been demonstrated include both monocots and dicots, for example Arabidopsis, carrot, maize, Oenothera, pea, Petunia and wheat. In fact, RNA editing in plant mitochondria appears to pre-date the divergence of the Pteridophyta (fernqike plants) and Spermatophyta (seed plants) and may thus be an ancient trait (R. Hiesel, B. Combettes and A. Brennicke, unpublished). The genomic sequence of the bryophyte Marchantia 9 suggests that RNA editing is not required in this moss. The rRNAs in higher plant mitochondria have been partially analysed by direct RNA sequencing in wheat 1° and by cDNA analysis in Oenothera n. Neither investigation revealed any definite indication of editing. One cDNA clone of the 26S rRNA in Oenothera mitochondria was found to differ from the genomic sequence at two positions11: one C to U and one reverse event located close together in a loop structure. Since these alterations were found in only one of several clones, their significance is unclear.
TIG SEPTEMBER 1 9 9 2 VOL. 8 NO. 9 ©1992 Elsevier Science Publishers Ltd (UK)
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VlEWS It is not known whether tRNAs are edited. Comparison of tRNA genes in different species shows C-to-U transitions at some positions that may be compensated for by RNA editing. Unfortunately, since direct sequencing of several tRNAs was done in species different from those for which genomic sequences are available, no clear deductions about editing in tRNAs can be made. All of the protein-coding mRNAs investigated in higher plant mitochondria are with the exception of T-urfl3- edited to some extent. The degree of editing, however, differs considerably for different genes and in different species. The most complete set of data is currently available for Oenothera mitochondria, where more than 20 genes have been investigated for RNA editing (Fig. 1). The nad3 mRNA is one of the most extensively edited transcripts, with 4.5% of all nucleotides altered in Oenothera 13 and 13% of the amino acids altered in wheat mitochondria 14. The least edited open reading frame is atpA in Oenothera, where only 0.25% of the nucleotides are edited n.
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The C-to-U alterations made by RNA editing will obviously affect only codons containing C residues. Most frequently FIGn altered are first and second codon positions RNA editing sites in Oenotheramitochondria are clustered in open reading that change the identity of the encoded frames. Each experimentally determined editing site is indicated by an arrow. amino acid. The functional importance of Closed boxes represent the open reading frames of individual genes and exons analysed for editing, dashed open boxes parts of genes not analysed. the less frequent silent changes at the third Solid lines represent noncoding sequences analysed for RNA editing. Exons codon position is as yet unclear. Of obvious importance are alterations creating AUG are marked in alphabetic order. Abbreviations: atpA, atp6, atp9, ATPase initiation codons, as in the had1 mRNA of subunits; cob, cytochrome b; coxI, coxlI, coxIII, cytochrome c oxidase subunits; mat-r, maturase-related reading frame; nadl, nad3, nad5, NADH wheaO 5 and in chloroplasts 4,5, and editing dehydrogenase subunits; orf, orfB, open reading frame; rps3, rpsl3, rpsl4, events resulting in termination codons. rpsl9, rpl5, ribosomal proteins of the small or large subunit; 5S rRNA, 18S Analysis of the atp9 gene product in wheat rRNA, 26S rRNA, ribosomal RNAs. mitochondria revealed that the UGA termination codon created by RNA editing resulted in a correspondingly shortened polypeptide ]6. in Oenothera, for example, is partially edited in several RNA editing has resolved the long-standing debate positions. The deduced translation products of both partially and fully edited mRNAs show similarity with about whether plant mitochondria have a nonstandard the corresponding proteins of several other species 9, genetic code. From genomic sequence comparisons, suggesting that in theory proteins synthesized from both CGG and the normal triplet UGG had been suspected to code for tryptophan 17As. Investigation of RNA both types of mRNAs could be functional. editing has shown that nearly all CGG codons at posThe only plant mitochondrially encoded polypeptides for which sequence data are available are the itions where tryptophan is evolutionarily conserved are altered to UGG. Thus there is no longer any need to amino-terminal portion of the coxIIgene product from sweet potato ]9, and the atp9 gene product from postulate a divergent genetic code in plant mitochonwheat16, 20. The former gives no indication of RNA editdria. ing, while the latter confirms all editing sites determined by cDNA analysis. There is no indication of hetProtein families by differential editing? erogeneity in the amino acid sequence of the atp9 For several plant mitochondrial genes, comparisons of independent cDNA clones show that transcripts of gene product, in agreement with the observation that the same gene may be edited to different degrees. If all editing sites in the wheat atp9 transcripts appear to be completely edited in the mRNA pool. In the case of all these mRNAs were equally accessible for transgenes for which a high percentage of partially edited lation, slightly different polypeptides could be synmRNAs has been observed, protein sequence data are thesized. The transcript population of the nad3 gene TIG SEPTEMBER 1 9 9 2 VOL. 8 NO. 9
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[]~EVIEWS NUCLEOTIDE REPLACEMENT
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FIG[] The biochemical reactions possible for RNA editing in plant mitochondria. (A) Deamination of a specific cytidine in the RNA chain catalysed by a cytidine deaminase activity. (B) Removal of the cytosine base by an RNA glycosylase activity without cleavage of the sugar-phosphate backbone and the subsequent insertion of an uracil base. (C) Nucleotide replacement by excision of a C and insertion of a U. The parts of the molecules affected by the potential reactions are circled.
OH OH 3'
required to resolve the question of w h e t h e r variant p o l y p e p t i d e s can be synthesized from a single gene by differential RNA editing.
How does RNA editing work in plant mitochondria? The biochemistry of RNA editing in plant mitochondria is as yet unclear. Three different reactions could theoretically be responsible for the C-to-U transitions: deamination, base substitution and nucleotide excision and insertion (Fig. 2). DNA deamination is a spontaneous reaction and a c o m p l e x repair system has .evolved to eliminate the errors introduced by it. Deamination of a C to U alters the sequence of the apolipoprotein B mRNA in the cytoplasm of some mammalian tissues, a reaction that
appears to be mediated by a single p o l y p e p t i d e 2122. Furthermore, cytidine m o n o n u c l e o t i d e deaminase activities have b e e n found in higher plants, but are not localized to any c o m p a r t m e n t and have not b e e n tested for their activity on polynucleotides. The rare reverse events might require a different enzymatic catalysis in the form of a CTP synthase activity. The close proximity of C-to-U and U-to-C editing events in a 26S rRNA cDNA clone in Oenothera could suggest a transamination mechanism that transferred the amino group from the C to the U residue in the same rRNA molecule. However, such a reaction w o u l d require n e w enzymatic activities and remains speculative. The second possibility is that editing could p r o c e e d via excision of the base without interrupting the
TIG SEPTEMBER1992 VOL. 8 NO. 9
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[]~EVlEWS
sugar-phosphate backbone. Such RNA glycosylation reactions have been described in tRNAs and it is plausible that they may also alter mRNA sequences in plant mitochondria. The third possible pathway involves a complete cut of the nucleotide chain, excision of the cytidine, addition of a uridine and subsequent religation of the interrupted molecule. Trypanosome mitochondria add and delete uridines in their mRNAs by such a mechanism, which involves transesterifications in the insertion and religation steps 23,24. However, numerous intermediates have been identified in trypanosome mitc,chondria in which the number of uridines deviates from the final sequence. No clear evidence for such aberrations has yet been observed in any of the completely or incompletely edited plant mitochondrial transcripts, even though large numbers of cDNA clones and sequences amplified by the polymerase chain reaction (PCR) have been investigated.
Which nucleotide is edited? The specificity of RNA editing in plant mitoch6hdria is probably determined independently from the biochemical activity. The many different editing sites without any overt similarity in the surrounding nucleotide sequences seem to require a complex mechanism for determining specificity. The editing event(s) in the apolipoprotein B mRNA are recognized solely in their sequence context by a protein factor22. In contrast, no common motifs can be discerned in the vicinity of RNA editing sites in plant mitochondria apart from a loose bias in nucleotide distribution, with guanosines found only rarely as the nucleotide preceding an edited cytidine25, 26. The species-specific editing events observed in identical homologous genes rule out the primary sequences immediately surrounding editing sites as the sole determinants of an editing site. Although some short sequence similarities have been identified between selected edited regions, no consensus sequence could be deduced that covered all known editing sites 7. RNA editing in plant mitochondria does not proceed in a single direction along the mRNA molecule, but rather appears to select the nucleotide that is to be edited by differential affinity to individual sites. Antisense RNA molecules unique for each site could provide the specificity of plant mitochondrial editing, analogous to the guide RNA model for trypanosome mitochondria 23,24. Guide RNA molecules for editing in plant mitochondria have not yet been identified although they have been looked for extensively. If they exist, their sequence specificity would not require more than nine or ten nucleotides for base pairing to allow unambiguous definition of a given editing site - and such short sequence similarities may be difficult to identify.
Are some RNA editing events mistakes? The identification of several RNA editing events that have no apparent advantage may provide some further clues to the specificity determinants of RNA editing in plant mitochondria. A pseudogene of rps19 in Oenothera mitochondria 27 is edited at positions differing from the editing sites of the functional open reading frame in Petunia mitochondria 2s. Only one
site is conserved, and this is a silent nucleotide position. Most of the other editing sites in the functional gene in Petunia improve the evolutionary conservation of the protein, while the editing events in the rpsl9 pseudogene in Oenothera lessen the degree of similarity but are nevertheless specific to certain cytidine residues. This finding suggests that the editing specificity can shift from one nucleotide position to other(s) if functional pressure is released. Apparent 'mistakes' in editing specificity have also been observed in essential coding regions in spite of a functional requirement for these proteins. Termination codons have been found after editing in the first few triplets of coxI, atp6and rps3 mRNAs in Oenothera mitochondria, presumably precluding synthesis of the encoded polypeptides 27. These 'mistakes' are as (in)frequent as some of the silent editing events, which might likewise originate from such a loose specificity, but may be tolerated because they do not affect the translation product. Introduction of translational stops by editing may on the other hand have some regulatory function in modulating the translatable RNA pool.
Editing in plant mitochondria is post-transcriptional The first indications that RNA editing in plant mitochondria was post-transcriptional came from analysis of individual cDNA clones from several protein-coding genes that were found to be differentially edited 13. Such partially edited molecules may represent editing intermediates that are further edited to the mature mRNA molecules. In maize and Petunia mitochondria x9,3°, spliced coxlI transcripts are more extensively edited than unspliced molecules. The large transcripts of nad3 in wheat - presumed to be the precursor molecules - are less extensively edited than a smaller transcript of this gene that may be a processing produc0 4. These observations suggest that RNA editing is a true posttranscriptional process. Cis
and trans splicing in plant mitochondria
In addition to RNA editing, complex splicing processes are required to create meaningful open reading frames for some plant mitochondrial polypeptides. The presence of introns was documented when the first plant mitochondrial gene was identified and sequenced 17. All higher plant mitochondrial introns that have been described show typical features of group II introns and can be folded into the consensus model structure. Group I introns have not yet been detected in these organelles. To date, about a dozen cis-splicing introns have been found in plant mitochondria and more are likely to turn up as new genes are identified (Table 1). The identification of genes is made particularly complicated by transsplicing events such as those recently found in wheat, maize, Petunia, Oenothera and Arabidopsis mitochondria 15,31,32 (Table 1). These trans-splicing events occur exclusively in the transcripts encoding subunits of the NADH dehydrogenase complex (Table 1). The complete open reading frame of the nadl gene is encoded by five exons, connected by two cis- and two trans-splicing events
TIG SEPTEMBER 1 9 9 2 VOL. 8 NO. 9
[]~EVIEWS
The plant mitochondrial trans-splicing events appear mostly to pre-date the divergence of at least monocots and dicots. Some trans-splicing events, however, must be of recent origin, since Tra~s Intron the wheat, Petunia and Oenothera open reading inttons ~ editing Ref. frames differ in their organization and in the ~ I (wheat/Petun/a) 1 3 ? 15.32 number of trans-splicing events (Fig. 4). The ~o bidopsts) 2 2 ÷/? 31a cis-spliced intron between exons d and e of nadl requires an additional trans-splicing event (~otbera) 3 1 + 35 in wheat and Petunia. These trans-splicing introns possibly result from the frequent recombinad4 (wheat/Arabidopsis) 3 ? 40~ nation within these plant mitochondrial genomes rearrangements that have disrupted the once had5 ( ~ idopsis) 2 2 ÷ 33 c/s-splicing introns without deleterious consequences as long as the open reading frame can t i o n s : - , no intron of the respective ~ present; +, intron e d i ~ g identified: ?, intron sequences not investigated. be assembled in trans. Gene ature is as m Fig. 1. The specificity of trans splicing is possibly ~W, Schuster and A. Brennicke, unpublished, associated with a slightly longer base-pairing stem of the interrupted domain IV. These pairings connect the two mRNA molecules to in Oenothera, but one cis- and three trans-splicing reconstitute the former cis-splicing intron that was events in wheat and Petunia. The nad5 transcripts in broken by genomic recombination 15,31. Experimental Oenothera, Arabidops/s, wheat and maize require two evidence is still required, however, to show that these cis- and two trans-splicing events to integrate an exon sequences are involved in the specificity of trans splicof only 22 nucleotides33,34. One trans-splicing reaction ing. It has been suggested that such split group II and three cis-splicing events are needed to connect the introns may represent the first steps in the evolution of open reading frame of the nad2 gene in Oenothera nuclear intron splicing, where most of the activities and Arabidops/s35. have been dissociated from the actual intron and n o w The trans-splicing reactions in plant mitochondria function in trans. appear to be associated with the presence of intron sequences that can be aligned by base pairing to form RNA editing in i n t r o n s an interrupted typical group II intron structure. The Intron splicing and RNA editing appear to be insecondary structure models place the discontinuity in dependent yet interdependent post-transcriptional prothe mitochondrial trans-splicing introns in domain IV cesses in plant mitochondria. Several intron sequences (Fig. 3). This location is different from the transappear to require RNA editing in order to fold into the splicing intron model of rps12 in higher plant chlorocorrect secondary structure, which is presumably plasts36, where the intron sequence is interrupted in essential for function. domain III, but is similar to the psaA transcripts in Two RNA editing events have been observed31 in Chlamydomonas chloroplasts37, where an intron is the 3' region of the trans-splicing intron a/b in the fragmented in domains I and IV. nadl mRNAs of Oenothera that appear to be essential for folding of domain VI. Other editing events in the stem regions of domain VI were found in a cis-splicing intron of nad5 domain III mRNA and in the trans-splieing nadl intron c/d; both of these events are also needed to maintain the conserved base-paired configuration31,33. The single trans-spliced intron sequence of the nad2 gene is edited once in domain I and once in the downstream stem part of domain IV (Ref. 35). The edited nucleotide in domain I appears to be indispensable in defining this stem and therefore to be essential for intron folding and excision. Both editing sites observed in the nad2 intron sequences are edited as frequently as non-silent editing sites in open v, reading frames, further supporting their functional importance. These intron editing events have so far only been determined in intron sequences of FIG[] unspliced precursor mRNAs. The complete Secondary structure models of the nucleotide sequences flanking trans-splicing exons in plant mitochondria suggest the involvement of group set of RNA editing sites required for a functional intron structure, however, can only be II intron sequences. These models localize the disruption in the structure model in domain IV, one of the variable regions of such introns. determined in the excised intron sequence.
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TIG SEPTEMBER 1 9 9 2 VOL. 8 NO. 9
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Whyeditingand splicing? In the conventional dogma pre-mRNA 2 'DNA makes RNA makes protein', there is no obvious place either for RNA editing "~. "~..~" ,~" or for introns. The arguments mature mRNA • I = Iblcl°l I invoked to rationalize the ..,¢ . f.,¢'~. '~. existence of introns (evol.." ...::::-'" ".. ".. utionary advantage, selfish ~-..-:2-';"" . """ pre-mRNA1 - - t a propagation, control of gene expression) must also be inpre-mRNA 3, voked for RNA editing. At mat.-r. present we can barely even speculate on any 'selfish' Petunia attribute of RNA editing, unless it perhaps confers an pre-mRNA 3 I-advantage on the organelle itself, protecting its rudipre-mRNA 4 mentary genome from slipping into the nucleus. Against this wheat suggestion, edited versions of lost or truncated mitopre-mRNA 3 [~-chondrial genes have been found to be encoded by nupre-mRNA 4 I mat.-r. clear genes, exemplified by the coxII gene in pea38 and the rpsl2 gene in Oenothera pre-mRNA 1 (L. Grohmann, A. Brennicke and W. Schuster, unpublished). , Less far-fetched may be a connection with regulatory mature mRNA --] a I mechanisms that could act b °41 ...... .... "% through RNA editing to alter the pool of translatable pre-mRNA 2 mRNAs. Several RNA editing events in plant mitochondria pre-mRNA 3, suggest just such a connection. I ° i The examples of RNA editing in intron sequences discussed FIG[F] in the previous section are the Top: Exons of the nadl genes in Oenotheraz:, Arabidopsis (W. Schuster, unpublished), most obvious candidates, since Petunia 32 and wheat 15 require several trans~plicing events for assembly of the open reading frame. The two trans~.plicing reactions between exons a/b and c/d are conserved their requirement for splicing in all species, whereas the Oenotbera and Arabidopsis cis-splicing intron between exons die could be used to regulate is interrupted in wheat and Petunia in different positions, resulting in an additional the levels of spliced, mature trans-splicing event in these plants. In the latter two species the mature nadl mRNAis transcripts. Another example thus assembled from four pre-mRNA molecules. Such trans-splicing events are probably of a potential regulatory funcresults of the recombination events that frequently rearrange plant mitochondrial genomes. tion for an editing event in Bottom: Assembly of the mature mRNAfor the had5 gene in Oenotbera and Arabidopsis a noncoding sequence could from three different pre-mRNAmolecules. This gene is organized similarly in wheat and be provision of an improved maize. Abbreviations are as in Fig. 1. ribosome-binding site upstream of rpsl4 (Ref. 39). The first editing event first triplets of coxI and rps3 open reading frames in the n a d l open reading frames creates an AUG could be examples of such regulation. codon, the only potential start codon in wheat 15. Presumably the unedited transcript cannot be transAcknowledgements lated, and RNA editing at this site would thus be the We thank Stefan Binder, Lutz Grohmann, Rudolf Hiesel, limiting step in production of a translatable mRNA Volker Knoop and Anita Marchfelder for discussions and unpublished data. Work in our laboratory was supported by molecule. grants from the Deutsche Forschungsgemeinschafl and the Thus RNA editing could, at least in some instances, Bundesministerium for Forschung. provide a rapid transition from a pool of steady-state unedited and untranslatable mRNA to mature mRNAs, References without any de novo synthesis of transcripts. RNA edit1 Covello, P.S. and Gray, M.W. (1989) Nature 341, 662466 ing may also be able to regulate the reverse step, 2 Gualberto, J.M. et al. (1989) Nature 341,660-662 making mRNAs untranslatable and condemning them 3 Hiesel, R., Wissinger, B., Schuster, W. anti Brennicke, A. to degradation. The termination codons found in the (1989) Science 246, 1632-1634 L
i
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4 Hoch, B. etal. (1991) Nature353, 178-180 5 Kudla, J. etal. (1992)EMBO.L 11, 1099-1103 6 Schuster, W., Hiesel, R., Wissinger, B. and Brennicke, A. (1990) Mol. Cell. Biol. 10, 2428-2431 7 Gualberto, J.M., Well, J-H. and Grienenberger, J-M. (1990) Nucleic Acids Res. 18, 3771-3776 8 Ward, G.C. and Levings, C.S., III (1991) PlantMol. Biol. 17, 1083-1088 9 0 d a , K. etal. (1992)J. Mol. Biol. 223, 1-7 10 Spencer, D.F., Schnare, M.N. and Gray, M.W. (1984) Proc. Natl Acad. Sci. USA 81,493-497 11 Schuster, W. et al. (1991) Curr. Genet. 20, 397-404 12 Welt, J-H. (1988) PlantMol. Biol. Rep. 6, 30-31 13 Schuster, W., Wissinger, B., Unseld, M. and Brennicke, A. (1990) EMBOJ. 9, 263-269 14 Gualberto, J.M., Bonnard, G., Lamattina, L. and Grienenberger, J-M. (1991) Plant Cell 3, 1109-1120 15 Chapdelaine, Y. and Bonen, L (1991) Cell 65, 465-472 16 Begu, D. el al. (1990) Plant Cell 2, 1283-1290 17 Fox, T.D. and Leaver, C.J. (1981) Cell26, 315-323 18 Hiesel, R. and Brennicke, A. (1983) EMBOJ 2, 2173-2178 I 9 Maeshima, M., Nakagawa, T. and Asahi, T. (1989) Plant Cell Physiol. 30, 1187-1188 20 Graves, P-V. et al. (1990) J. Mol. Biol. 214, 1-6 21 Powell, L.M. etal. (1987) Cell50, 831~840 22 Greeve, J., Navaratnam, N. and Scott, J. (1991) Nucleic Acids Res. 13, 3569-3576 23 Blum, B., Bakalara, N. and Simpson, M. (1990) Cell 57, 355-366 24 Blum, B., Sturm, N.R., Simpson, A.M. and Simpson, L. (1991) Cell65, 543-550
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Chickens in search of a market
Genetics and Evolution of the Domestic Fowl by Lewis Stevens Cambridge University Press, 1991. £50, $89.95 (xiii + 306 pages) ISBN 0 521 40317 0 Most genetics textbooks focus on the subject itself, drawing examples from whichever taxonomic group can best exemplify the point under consideration. Occasionally, however, a book appears that is restricted to a particular species one that attempts to discuss the current state of the art (sorry, science) for that organism. This is just such an attempt, and is a splendid little book that is the first devoted to the domestic fowl for many years. Because of its very nature, Genetics and Evolution of the Domestic Fowl covers a wide range of topics. The opening chapter discusses the evolutionary history of the species, and describes the contribution of genetics to its elucidation. The results support the general consensus from anatomy and behaviour that the closest relative of the domestic fowl is probably the Red Jungle Fowl (Gallus gallus), but the possibility of a polyphyletic origirr cannot be ruled out. There then follow chapters on genetic principles, cell biology, mendelian inheritance, sex
linkage and gene mapping to set the scene for the more detailed sections on anatomical genetics - feathering, plumage variation, nerves, musculature, etc. A very good chapter on quantitative genetics shows how the poultry industry has applied selection and environmental management to improve the product out of all recognition in the past 20 years. The book then moves on to the really technical stuff- protein evolution, immunogenetics, gene cloning and transfer, and a final appendix on oncogenes. As I said, an excellent little book, but who will buy it? If they have any sense, commercial breeders will hire a professional geneticist to help them through the morass of responses to selection, immunization procedures, and so on. But there is also a whole subculture out there of folk who enjoy keeping and breeding chickens for pleasure and show. Might they be persuaded to buy? I doubt it. I have some knowledge of a parallel group who keep pedigree dogs, and its T1G SEPTEMBER 1 9 9 2 VOL. 8 NO. 9
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aficionados would find a book on dogs pitched at an equivalent level very hea W going. The sections on basic inheritance and linkage would be useful, but a little more on the differentiation of breeds would probably have been welcome to this rather specialist, if nontechnical, market. The chapter on quantitative genetics would be an excellent basis for a short course in animal improvement, but it is perhaps expecting a great deal of a student to buy the book for a single chapter. The later sections are also excellent reviews of those aspects of molecular genetics relevant to birds in general, and fowls in particular. I think, though, that the background theory is equally well presented in more general texts. So I wonder where the market lies? Despite these reservations, I really enjoyed this book, and will refer to it frequently. My interest in birds has not extended to their immunology or oncogenetics, and it fills that gap to perfection. I will also use the chapter on quantitative genetics in my teaching, so it will not gather dust on the shelf as so many other books do.
David T. Parkin Department of Genetics, School of Medicine, Queen's Medical Centre, Nottingham, UKNG72UH.
