RNA editing

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RNA editing Axel Brennicke *, Anita Marchfelder, Stefan Binder Allgemeine Botanik, Universita«t Ulm, D-89069 Ulm, Germany Received 14 October 1998; revised 11 December 1998; accepted 14 December 1998

Abstract The term RNA editing describes those molecular processes in which the information content is altered in an RNA molecule. To date such changes have been observed in tRNA, rRNA and mRNA molecules of eukaryotes, but not prokaryotes. The demonstration of RNA editing in prokaryotes may only be a matter of time, considering the range of species in which the various RNA editing processes have been found. RNA editing occurs in the nucleus, as well as in mitochondria and plastids, which are thought to have evolved from prokaryotic-like endosymbionts. Most of the RNA editing processes, however, appear to be evolutionarily recent acquisitions that arose independently. The diversity of RNA editing mechanisms includes nucleoside modifications such as C to U and A to I deaminations, as well as non-templated nucleotide additions and insertions. RNA editing in mRNAs effectively alters the amino acid sequence of the encoded protein so that it differs from that predicted by the genomic DNA sequence. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Contents 1. 2. 3.

Introduction ^ no border between RNA editing and RNA modi¢cation . . . . . . Di¡erent species ^ di¡erent genomes ^ di¡erent organelles ^ di¡erent processes RNA editing in the nucleus of animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. RNA editing in vertebrate apolipoprotein B mRNAs . . . . . . . . . . . . . . . 3.2. RNA editing in glutamate-responsive ion channels . . . . . . . . . . . . . . . . . 4. RNA editing in kinetoplasts of trypanosomes . . . . . . . . . . . . . . . . . . . . . . . . . 5. RNA editing in mitochondria of Acanthamoeba . . . . . . . . . . . . . . . . . . . . . . . . 6. RNA editing in mitochondria of Physarum . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. RNA editing in mitochondria and plastids of plants . . . . . . . . . . . . . . . . . . . . 8. RNA editing in mitochondria of animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Cotranscriptional RNA editing of virus RNAs . . . . . . . . . . . . . . . . . . . . . . . . 10. RNA editing and its consequences and advantages . . . . . . . . . . . . . . . . . . . . . 11. Origin and evolution of RNA editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Conclusion and recommendation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Tel.: +49 (731) 50-22610; Fax: +49 (731) 50-22626; E-mail: [email protected] 0168-6445 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 4 5 ( 9 9 ) 0 0 0 0 9 - 1

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction ^ no border between RNA editing and RNA modi¢cation RNA editing processes are con¢ned to the world of eukaryotes and have not so far been recognized in microorganisms. Just as introns were eventually identi¢ed in bacterial genes including some in Escherichia coli, posttranscriptional changes in RNA sequences described as RNA editing may also occur in prokaryotes. RNA editing covers a multitude of biochemically di¡erent processes, the term having been adopted in 1986 for the addition and deletion of uridine nucleotides to and from mRNAs in trypanosome mitochondria [1]. The observed changes in nucleotide identity or insertion/excision of a nucleoside described below are termed RNA editing rather than being classi¢ed as the classical (30 years older) `RNA modi¢cation'. The latter processes change (modify) the nucleoside in the RNA chain, sometimes only by a minor modi¢cation, for example by the addition a methyl group, at other times signi¢cantly (Fig. 1) [2]. Sometimes modi¢cation even alters the nucleotide structure to such an extent that base-pairing properties change signi¢cantly [3]. These modi¢cations are most prevalent in tRNAs and rRNAs and are often essential for biological function. In some tRNAs a cytidine in the anticodon can be modi¢ed to function as a uridine in codon recognition to read di¡erent nucleotide triplets in the mRNA. In some of these

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cases modi¢cations in both eukaryotes and bacteria change the information content of the tRNA so that it does not faithfully re£ect the genomic blueprint. A further complication includes modi¢cation of some tRNAs by 3P additions of oligoA, which however is commonly described as RNA editing. Here we adopt the de¢nition of Price and Gray [4], which states that RNA editing describes processes of nucleotide alterations which result in di¡erent or additional nucleotides in the RNA which could as well have been encoded in the genomic sequence.

2. Di¡erent species ^ di¡erent genomes ^ di¡erent organelles ^ di¡erent processes RNA editing is found in very diverse species of eukaryotes, in humans and trypanosomes, in slime molds and plants (Table 1). The evolutionary distances between the di¡erent organisms in which editing has been observed not only re£ect the distribution of researchers' favorite organisms, but also show the wide evolutionary distances between the di¡erent editing mechanisms. While one level of classi¢cation can thus be based upon a phylogenetic scheme, another grouping could re£ect the mechanisms involved. In `insertional editing' nucleotides are inserted into (or deleted from) the single stranded RNA molecule. These insertions are not speci¢ed in the gene se-

C

a

Insertion/deletion editing is de¢ned as an editing process during which phosphodiester bonds are made and/or broken, resulting in almost all cases in an edited RNA in which the number of nucleotides has changed. In modi¢cation editing, the identity of a nucleotide is changed by base conversion or substitution without disruption of the phosphodiester backbone. The status of G to A and U to A editing in two mammalian RNAs is unclear: it could be the result of either base substitution (in which case it would qualify as modi¢cation editing) or nucleotide replacement (in which case it should be considered insertion/deletion editing). b For modi¢cation editing, the translational consequences of editing have been indicated for RNAs that contain a single editing site. c For other references, please check the corresponding chapters. d In platypuses, As and Cs are added. e A comparison of genomic and cDNA sequences shows multiple apparent A to G changes, suggesting that the RNAs are edited by A to I conversion (see chapter 19 of [55]). The presence of Is in edited RNAs has not yet been established, however. f Reprinted from [55] with kind permission.

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Table 1 The di¡erent processes of RNA editingf Typea

Organism (genome) Transcript(s)b

Insertion/deletion editing U insertion/deletion Kinetoplastids (mt) mRNAs

cis-acting element(s)

trans-acting factor(s)

Mechanism

Anchoring sequence

Cleavage, TUTase or U exonuclease action, and ligation

Physarum mRNAs, tRNAs, rRNA polycephalum (mt)

?

gRNAs, TUTase, RNA ligase, endonuclease, U exonuclease, other factors ?

P mRNA

Slippery sequence

Viral polymerase

A insertion

Paramyxoviruses (v) Ebola viruses (v)

GP mRNA

Slippery sequence

3P-terminal A addition

Vertebrates (mt)

mRNAs

Flanking tRNA structure 3P-overlapping tRNA ? Internal guide sequence ?

Mostly C insertion ; also U, UA, AA, CU, GU, and GC G insertion

3P-terminal A additiond Metazoan animals tRNAs (mt) C to A, A to G, U to Acanthamoeba tRNAs G, and U to A castellanii, chytridiomycete fungi (mt) Modi¢cation editing C to U Land plants mRNAs (mt, cp), tRNAs (mt), rRNAs (mt) Mammals (n) mRNAs, apoB (GlnCstop), NF1 (ArgCstop) Physarum cox1 mRNA polycephalum (mt) Marsupials (mt) tRNA (GlyCAsp anticodon) U to C Land plants (mt, mRNAs cp) Mammals (n) WT1 mRNA (LeuCPro) A to I Mammals (n) mRNAs, GluR-B, -C, -D, -5, -6, 5-HT2C R Human hepatitis Antigenome (stopCTrp) delta Squids (n) Kv2 K‡ channel mRNA A to I?e Drosophila 4f-rnp mRNA melanogaster (n) Mice (n) GPT mRNA (CysCTyr) G to Ac U to A

Humans (n)

K-Galactosidase mRNA (PheCTyr)

Linked to transcription

Pseudotemplated transcription Viral polymerase Pseudotemplated transcription Endonuclease, Cleavage and TATase TATase action Endonuclease, Cleavage and TATase TATase ? action Endo- or exonuclease, Replacement of ¢rst nucleotidyltransferase ? three 5P nt

Flanking sequence ?

C deamination

Mooring sequence, e¤ciency and AUrich elements ?

C deaminase (APOBEC-1), other factors ?

C deamination

?

?

?

Flanking sequence ? ? dsRNA structure

?

U amination ?

dsRNA structure

? ? dsRNA A deaminases A deamination (ADAR1 and -2) ADAR1 A deamination

? ?

? ?

A deamination? A deamination?

?

?

?

?

Base or nt replacement ? Base or nt replacement ?

ADAR, a deaminase for RNA; apoB, apolipoprotein B; cox1, cytochrome c oxidase subunit 1; GluR, glutamate receptor ; GP, glycoprotein; GPT, GlcNac-1-phosphate transferase; 5-HT2C R, serotonin 2C receptor ; NF1, neuro¢bromatosis type 1 gene; TATase, terminal uridylyltransferase ; WT1, Wilms' susceptibility gene 1; cp, chloroplast; ds, double stranded; mt, mitochondrial; n, nuclear ; nt, nucleotide; v, viral; ?, uncertain or unknown. This (putative) editing event has been reported for the NIH/SW mouse strain. Whether it occurs in other strains and species is unknown.

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Fig. 1. Phylogenetic distribution of various types of nucleoside modi¢cations in RNA. Reprinted from [55] with kind permission.

quence from which the RNA has been transcribed. In the insertional editing processes found in mitochondria of the Trypanosoma parasites and of the slime mold Physarum, the mature mRNA will thus be longer than the respective primary transcript [5^8]. In trypanosomes some mature mRNAs have more nucleotides inserted than are encoded by the gene. In these cases the translated mRNA may thus be more than twice as long as the actual gene in the DNA. Insertional RNA editing also includes the nontemplated cotranscriptional insertion of nucleotides in certain viruses, which is speci¢ed by the surrounding sequence [9]. Other borderline cases are the 3P polyA additions which complete UAA translational stop codons and some tRNAs in animal mitochondria. A second class of RNA editing, `modi¢cation editing', is more closely related to the RNA modi¢cation processes as re£ected in the term. In this category the two most prevalent changes induce deaminations of C to U and A to I. Less widely distributed changes

are U to C, G to A and U to A conversions, the latter being found in mammals such as mice and humans. To summarize the details of RNA editing processes from the clearly eukaryotic nuclear-cytoplasmic editing events to the RNA editing processes found in the organelles, we will require a few novel concepts developed in the investigation and description of RNA editing. One of these is the term `editosome', which describes the usually unknown protein or protein-RNA complex responsible for the RNA editing reaction. Another concept is the `guide RNA' molecule, which through base-pairing with the RNA molecule to be edited speci¢es the editing site. An editing site is the nucleotide actually altered and can include those nucleotides immediately adjacent or in the immediate vicinity.

3. RNA editing in the nucleus of animals RNA editing of transcripts of nuclear genes in animals are very rare events and have been reported

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for only three or four speci¢c genes. Examples in mammals are listed in Table 1 and include the single C to U changes in the apolipoprotein B and neuro¢bromatosis type 1 tumor suppressor mRNAs, a U to C alteration in Wilms' tumor susceptibility mRNA, A to I deaminations in GluR channel mRNAs and in 5-HT2C R serotonin receptor mRNAs. A to I changes have also been described in Kv2 K‡ channel mRNAs from squid, in the Drosophila 4f-rnp mRNA and in the human hepatitis delta virus. G to A changes have been observed in the mouse GlcNac-1 phosphate transferase mRNA, and U to A alterations in human K-galactosidase mRNAs. The nucleotide speci¢cities and the enzymatic mechanisms involved in the tissue-speci¢c C to U editing in the apolipoprotein B and the A to I deaminations in GluR channnel mRNAs have been characterized and will be considered in more detail [10,11]. 3.1. RNA editing in vertebrate apolipoprotein B mRNAs In vertebrates a single apolipoprotein B (apoB) gene (Fig. 2A) encodes two di¡erent proteins, one of 100 kDa (APOB100) and the other of 48 kDa (APOB48) [11^18]. In humans APOB100 is synthesized in the liver, whereas APOB48 is made in the small intestine. Both proteins are involved in binding lipids to keep them in solution. APOB100 is secreted into the blood plasma, while APOB48 is involved in binding and resorbing fatty acids in the lining of the small intestine. In this tissue the apoB mRNA is modi¢ed at nucleotide 6666, where a cytosine is deaminated to a uridine. The CAA codon is converted to a UAA translational stop codon and as a result protein synthesis terminates to yield the smaller APOB48. This deamination reaction is catalyzed by a sitespeci¢c enzyme, the APOBEC-1 enzyme, which will also deaminate cytidines in vitro [19]. This is the catalytic as well as the sequence-speci¢c subunit of an `editosome' complex, which contains at least three other as yet unidenti¢ed proteins. These are required for the highly speci¢c activity of apobec-1 in vitro, particularly to bind and access the apoB mRNA. In the yeast two-hybrid system an APOBEC-1 binding protein has been identi¢ed, which shows speci¢c af-

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¢nity for both the deaminase and the apoB mRNA. The tissue-speci¢c expression of the deaminase APOBEC-1 coincides with the distribution of the apoB RNA editing in di¡erent vertebrates. In humans and rabbits RNA editing and APOBEC-1 activity only occur in the small intestine, while in rats and mice both activities are also present in the liver. In addition to liver and small intestine the APOBEC-1 activity is expressed in some tissues where the apoB gene is not activated. In these cells APOBEC-1 may play a role in the deamination of other mRNAs, e.g. the nf1 (neuro¢bromatosis type 1 tumor suppressor) and the nat1 (novel apobec-1 target) mRNAs. The biological signi¢cance of the apoB mRNA editing and of the additional deaminations is as yet unclear, but may be revealed by experiments with apobec-1 overexpression and transient transfection experiments in knockout mice. These investigations have so far given equivocal results regarding the effects and importance of apoB editing. Transient expression of the APOBEC-1 deaminase in the liver by transfection reduces the amount of APOB100 with a corresponding increase of the APOB48 protein. After several days, however, the plasma concentration of the latter decreases. Overexpressing apobec-1 in mice in which the gene for the low density lipoprotein has been knocked out similarly decreases the concentration of APOB100 in the plasma and in e¡ect also lowers the concentrations of the APOB100 containing lipoproteins. Elevated cholesterol concentration based on misregulation of these two carriers thus appears to be in£uenced by the increased editing e¤ciency. Transgenic mice overexpressing apobec-1 developed carcinomas apparently due to the deleterious over-editing of other transcripts such as nf1 and nat1. In summary, RNA editing of a unique nucleotide in the apoB transcript results in the synthesis of two di¡erent proteins with di¡erent properties from one gene (Fig. 2A). In this case RNA editing increases product complexity from just one genomic coding region. However, considering the vast amount of non-coding DNA in the mammalian genome, encoding an additional gene does not seem to pose a problem. The presumed phenotypic and evolutionary advantage of this editing must thus be sought elsewhere, perhaps in the capability of a more rapid response to metabolic changes in the quality and

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quantity of fatty acids which are released depending on the type of food entering the small intestine. 3.2. RNA editing in glutamate-responsive ion channels In mRNAs encoding some of the subunits of glutamate-responsive ion channels (GluR), nucleotides recognized as G-residues were found at some positions, where A nucleotides were encoded by the respective gene [10,12,20]. These nucleotides turned out to be inosines (I) derived by hydrolytic deamination of the purine ring in the speci¢c adenosines. The I residues are recognized as G in almost all biological systems, including translation. This biochemical alteration of a nucleoside results in an RNA editing

event leading to an altered information content which directs synthesis of one or more di¡erent proteins. Such A to I modi¢cations are found in the di¡erent GluR subunit mRNAs and, depending on the subunit type, changes range from one to seven nucleotides (Fig. 2B). In addition, in the hepatitis delta virus RNA one and in a serotonin receptor transcript (5-HT2C R) four nucleotides are altered from A to I. Speci¢city in these RNA editing events is mediated by the preference of the single subunit deaminase (dsRAD or DRADA or ADAR1 = adenosine deaminase acting on RNA) for double stranded RNA [19,21]. Backfolding into extensive hairpin loops generates double stranded regions surrounding the nucleotide to be altered. These second-

Fig. 2. RNA editing in the nucleus of mammalia. A: RNA editing in the apoB transcript produces a stop codon by C deamination in a genomic CAA codon to a UAA codon in the intestine, but not in the liver. Two di¡erent proteins are synthesized from just one gene, one of 100 kDa as speci¢ed by the 29 exons of the genomic sequence (top part) and another of 48 kDa after editing in the intestine. B: Secondary structures folded between intron and exon sequences determines the A to I conversion sites in RNA editing of various glutamate-responsive channels (GluR), a serotonin receptor 5-HT2C R and the hepatitis delta virus (HDV). These A deamination editing sites are often found in double stranded regions of the primary transcript RNA. Reprinted from [55] with kind permission.

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Fig. 2 (continued).

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ary structures bind the enzyme and direct the activity to exposed adenosines. The ¢rst enzyme of the diverse family of dsRNA deaminases was identi¢ed by its destabilizing e¡ect on RNA duplexes before RNA editing was recognized. Base-pairings are destabilized by changing adenosines to inosines, the RNA duplex unwinds and becomes single stranded. In some gluR transcripts stable duplexes are only present in unspliced precursors, where intron and exon sequences can base-pair. This observation con¢rms that the A to I type of RNA editing occurs in the nucleus and not in the cytoplasm of animal cells, since it precedes intron splicing which only occurs in the nucleus. The physiological and phenotypic consequences of these editing events can be observed in the properties of the resulting ion channels. Depending on the respective proportion of edited and unedited subunits assembled in the multi-protein complexes which form the L-glutamate receptors, the properties of these main ion channels of excitatory synaptic signal transmission in the brain vary considerably. Coupled with the parallel alternative intron splicing of the precursor transcripts the varying extent of RNA editing in individual ¢nally translated mRNAs generates a range of combinatory possibilities resulting in synaptic ion channels with di¡erent properties. As in the case of the apoB mRNA editing, A to I editing in the nucleus of mammalia thus creates different protein products from a single gene. Again, an additional, more subtle level of regulation may be an underlying function of the posttranscriptional change in the resulting protein sequence. It is possible that RNA editing in cells of the nervous system can be in£uenced by developmental stimuli to yield a modulation of the ion channeling patterns during formation and maturation of the neuronal capacity of the brain.

4. RNA editing in kinetoplasts of trypanosomes When Rob Benne and his coworkers in Amsterdam claimed in 1986 that the mRNA for subunit 2 of the cytochrome c oxidase (cox2) in mitochondria of trypanosomes contains four U nucleotides which are not encoded in the mitochondrial genome, the scienti¢c community was very sceptical [1]. They

called the unknown process RNA editing, by analogy to the proofreading performed by the editors of manuscripts. The extent of editing in a mitochondrial transcript in Trypanosoma brucei, the causal agent of sleeping sickness, can be dramatic where more than half of the mature mRNA is posttranscriptionally inserted. This is for example observed in the transcript for subunit 3 of the cytochrome c oxidase (cox3), where the genomic coding region is 463 nucleotides long, and to which 547 nucleotides (all Us) are added and from which 41 of the genome encoded Us are deleted. The resulting edited mRNA is 969 nucleotides long and is translated into a conserved COX3 polypeptide. In the £agellated protozoa of the order Kinetoplastida, this type of RNA editing which inserts and deletes uridines is prevalent, and at the same time restricted to this class of organisms [5^7,22^ 27]. These protozoa are characterized by a single mitochondrion in the cell, the kinetoplast, which is located at the base of the £agella and thus presumably in an ideal position to provide the chemical energy for kinetic propulsion. In the kinetoplastid mitochondrial genome a classic set of genes encodes some of the hydrophobic subunits of the respiratory electron transport chain (Fig. 3). These genes are sometimes hardly identi¢able from their primary sequence conservation in the genome, particularly in those instances where extensive editing is required for the conserved open reading frame. In the nad9 transcript, encoding subunit 9 of the respiratory chain NADH dehydrogenase in T. brucei, 345 uridine nucleotides are added to create the 649 nucleotides long mRNA. Other primary transcripts such as nad4 and nad5 are not edited at all, the ¢rst identi¢ed RNA editing in cox2 (encoding subunit 2 of the cytochrome c oxidase) involves editing at three sites, where altogether four uridines are inserted. The ¢nal length of the mRNA encoding ATP6 (subunit 6 of the F0 -ATPase) of 811 nucleotides contains more uridines added by RNA editing (448) than the 392 nucleotides encoded by the mitochondrial genome. Trypanosomes have evolved a complex system to ensure the ¢delity of the number of nucleotides which have to be inserted as well as the precise location of the editing events in the transcript. In trypanosomatid RNA editing the location of the correct insertion site and the precise number of

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Fig. 3. RNA editing in trypanosome mitochondria involves the insertion and deletion of uridines into and from the primary transcript to create the mature, translated mRNA. A: Genes are encoded in the maxicircle molecules and require varying numbers of changes by RNA editing. The sites of U insertion and their number are speci¢ed by the guide RNAs (gRNA) encoded in the minicircle DNAs and transcribed from their own promoters located within 18-bp repeats. B: The guide RNAs base-pair with the transcript before editing downstream of a site of U insertions and anchor the guide to the transcript. The antisense sequence of the guide determines where and how many uridines are to be inserted or deleted. The mature edited mRNA can perfectly base-pair with the guide RNA. Reprinted from [55] with kind permission.

inserted Us are speci¢ed by the so-called `guide RNAs'. These are small RNA molecules, which contain a short antisense sequence which can base-pair with the unedited primary transcript downstream of

a site where uridines are to be inserted (Fig. 3B). As a result of such base-pairing the guide RNA is anchored to the transcript, the anchor forming a perfect match of 10^15 nucleotides between guide RNA

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and transcript RNA. This double stranded region and the speci¢c structure of the guide RNA are enveloped in a large multi-protein complex, the `editosome', which performs the actual editing. This editosome contains several enzymatic activities, which begin to open the transcript RNA chain at the ¢rst mismatched nucleotide and start inserting uridines. These will base-pair with the next nucleotides in the guide RNA and U insertion in the transcript will continue as long as A (or G) in the guide RNA is matched. Insertion of U into the transcript RNA molecule will stop when C or U is encountered in the guide, which can base-pair with the next original nucleotide in the transcript RNA. This mechanism requires certain properties of the editing process. Firstly, insertion or deletion of Us progresses from the 3P to the 5P end of the primary RNA molecule. Secondly, a single guide RNA will not cover all of the editing sites in an extensively altered primary transcript and several guide molecules will be required. Thirdly, in frequently edited transcripts an anchor site for a guide RNA in the 5P region of the transcript will sometimes only be created by the downstream editing events, thus resulting in a chain of guiding events proceeding in the 3P to 5P direction along the primary transcript eventually generating the mature mRNA. The guide RNA molecules are usually encoded in the so-called minicircle DNA (Fig. 3A), speci¢c molecules of the mitochondrial genome, of which thousands are present in a kinetoplast. They are mostly interconnected from unresolved replication catenates and form a type of three-dimensional `chain-mail' structure in the organelle. The guide RNA genes are characterized by a speci¢c sequence, which is present as an 18 nucleotides long imperfect repeat upstream of each guide RNA gene. These sequences possibly act as transcription promoters and may also play a role in the guide RNA evolution by duplication. The 18 bp long imperfect repeats are potentially involved in signaling individual transcript initiation, since most of the guide RNA molecules contain the di- or triphosphates characteristic for primary 5P termini. At their 3P ends guide RNAs have a tract of about 15 uridines which are added posttranscriptionally by a terminal uridylyl transferase. It has been proposed that these U tails act as potential donors of the Us inserted into the RNA during editing, but

experimental evidence suggests that UTP is utilized for the mRNA editing, possibly catalyzed by the same enzyme which adds the 3P oligo-U tails to the guide RNAs. The mechanism of U insertion and deletion involves an endonucleolytic cut at the mismatch between guide RNA and unedited transcript. The newly opened ends of the transcribed molecule are held in place within the editosome complex by several of the polypeptides contained therein. One of the enzymes in the editosome is a terminal U-transferase which adds Us from UTP in the 5P to 3P direction at the 3P end of the upstream part of the mRNA. Another enzyme, an RNA ligase, joins the two parts of the mRNA molecule after the gRNAspeci¢ed number of Us has been inserted. Probably during this alignment step the super£uous added Us as well as excessive genomically encoded Us are excised from the 3P end of the upstream part of the mRNA. This is where deletion of Us is templated by the overriding antisense sequence information of the guide RNA molecule. RNA editing of at least some of the mitochondrial mRNAs in these sometimes parasitic protozoa is developmentally regulated. Transcripts for cytochrome b and for subunit 2 of the cytochrome c oxidase (cox2) of T. brucei and T. congolese exhibit little or no editing in the bloodstream forms in the infected human or animal, where the respiratory chain is not functional in the parasites. These transcripts are only fully edited in the intermediate developmental forms, which are initiated in the midgut of the insect vector such as the tsetse £y, where the parasites depend on the ATP generated by the respiratory chain. RNA editing may thus confer a regulatory advantage if a clear chain of cause and consequence can indeed be established in this case. Minimizing the size of the mitochondrial genome cannot be the raison d'etre of RNA editing in trypanosomes, since the numerous guide RNAs take up considerably more genomic sequence than a normal protein coding gene.

5. RNA editing in mitochondria of Acanthamoeba RNA editing in the amoeboid protozoon Acanthamoeba castellanii only a¡ects tRNAs, where in addition a plethora of posttranscriptional modi¢cations

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Fig. 4. RNA editing in tRNAs. A: In Acanthamoeba nucleotides 1^3 (¢lled circles) often do not pair with the respective opposite nucleotides in the aminoacyl stem of many mitochondrial tRNAs. These nucleotides are removed and replaced with the correctly pairing nucleotides. B: In Physarum mitochondria nucleotides are inserted at various di¡erent positions in the tRNA precursor molecules by an as yet unknown speci¢city and mechanism. C: In metazoan animals missing or not base-pairing nucleotides at the 3P termini of tRNAs are added by the general mitochondrial polyA polymerase and the tRNA is completed at this end by the CCA adding enzyme. In marsupials a C nucleoside in the anticodon of tRNA-Asp is deaminated to a U, changing its identity to a tRNA-Gly. D: RNA editing in land plants alters tRNA sequences by C to U deamination mostly in basepairing regions and improves the secondary structure. The folding opposite strand may be involved in specifying these editing events. Reprinted from [55] with kind permission.

change numerous nucleoside structure(s) (Figs. 1 and 4). Following the de¢nition of RNA editing given above as nucleotide alterations which could just as well have been encoded in the genomic sequence, the sequence alterations in tRNAs of Acanthamoeba classify as bona ¢de editing [28,29]. During sequence

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analysis of the mitochondrial genome in this amoeba 13 of the 16 tRNAs encoded in the mitochondrial DNA were predicted to contain mismatches in the aminoacyl acceptor stem when folded according to the classical structure model. The mismatches occur exclusively in the ¢rst three base pairs, involving nucleotides 1^3 and the complementing nucleotides 70^ 72 respectively. These base-pairings are present in all tRNAs and are essential for the functional folding of the molecule in space and in some cases are crucial for aminoacylation. Direct sequence analysis of the mature tRNA molecules revealed that the nucleotides in positions 1^3 di¡ered from those predicted by the genomic sequence and that all of the mismatches in base-pairing to nucleotides 70^72 had been resolved. These posttranscriptional changes are limited to the ¢rst three nucleotides in all 13 tRNAs. G-U pairings are corrected to A-U pairs at nucleotide positions 1, 2 or 3, while a U-G low af¢nity pair involving nucleotide 4 is not altered. To change one or all of the ¢rst three nucleotides, replacement is the most parsimonious explanation, since a single biochemical modi¢cation reaction cannot result in the many di¡erent changes of nucleotide identity. One of the two enzymes required would be a tRNA-speci¢c 5P to 3P exonuclease, the other a 3P to 5P nucleotidyltransferase, which uses the 3P-terminal nucleotides of the tRNA as template to add only complementing nucleotides. The enzymes most likely to be involved have, however, not yet been identi¢ed or puri¢ed. The same pattern of tRNA editing as in Acanthamoeba mitochondria is also found in the mitochondria of some of the chytridiomycete fungi such as Spizellomyces punctatus [30]. These fungi are phylogenetically very distant and unrelated to Acanthamoeba, making a common evolutionary origin of this type of tRNA editing very unlikely. Thus apparently independent events have established the same patterns of RNA editing in amoebas and these fungi, suggesting that pre-existing enzymes could fairly easily be adapted and modi¢ed during evolution.

6. RNA editing in mitochondria of Physarum In the myxomycete slime mold Physarum polyce-

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Fig. 5. RNA editing in mitochondria of the slime mold Physarum. A continuous open reading frame, here for the cox1 subunit of the cytochrome c oxidase, is only created by the insertion of various nucleotides, mostly C, into the primary transcript. In addition several C to U deaminations are observed, which improve the conservation of the encoded protein with its homologues in other organisms. The depicted three reading frames show the resulting polypeptide sequence (underlined) to be derived as a continuous frame only in the edited mRNA. Reprinted from [55] with kind permission.

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phalum RNA editing appears to be the most complex described to date and involves both types of RNA editing, the insertional and the substitutional types [8,31,32] (Fig. 5). Any nucleotide, A, C, G or U, can be inserted as a monomer or as a dinucleotide, and in addition, C to U exchanges are observed. Nucleotide insertions are found in almost all of the RNA transcripts analyzed, all in all about 1000 editing sites are expected in the tRNA, rRNA and protein coding transcripts of the about 60-kb large mitochondrial genome. The insertion of nucleotides into transcripts in Physarum mitochondria is closely linked to the transcription processivity and is apparently very e¤cient, whereas substitutional editing occurs more slowly and by an unrelated mechanisms. Possibly the latter C to U transition is catalyzed by a deamination reaction analogous to the apoB RNA editing, since deletion/excision of a nucleotide has never been observed in this system. Insertional maturation of the newly synthesized RNA molecule is already complete 14^20 nucleotides away from the site of chain elongation by the RNA polymerase. Insertion probably utilizes nucleotide triphosphates, since speci¢c concentrations of CTP are required for the insertion of Cs at the proper sites. The hypothetical `editosome' is most likely linked to the transcription complex and only acts on nascent RNAs. Partial in vitro editing systems cannot insert the missing nucleotides posttranscriptionally into an unedited RNA molecule. The close coupling to transcription results in a 5P to 3P processivity of RNA editing, and thus contrasts with the 3P to 5P progression of the U insertions in trypanosome mitochondria. The close physical and temporal coupling of RNA polymerase activity and RNA editing in this system suggests an enzymatic involvement of the RNA polymerase itself in the editing process. Mitochondrial RNA polymerases are evolutionarily related to the prokaryotic phage T7 single subunit polymerase type, which has been found to add faithfully those nucleotides that are inserted during RNA editing in the Ebola virus transcript (see below). Thus some conserved sequence motif structure and additional speci¢city factors may cause the RNA polymerase to pause or even to `back up' and by re-reading the last transcribed nucleotide to insert the correct

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additional nucleotide, the polymerase thus stuttering. In this model the RNA polymerase has to reattach to the DNA template to resume faithful transcription and to then copy correctly at least up to the next editing site. The signal motif(s) for speci¢city in Physarum mitochondrial editing are as yet unclear, but clearly do not involve stuttering of the RNA polymerase. Firstly the site of nucleotide insertion has to be marked and secondly the type of nucleotide inserted must be identi¢ed. The physical connection of nascent transcript elongation and RNA editing suggests that in the elongating RNA the sequences 3P to the adjacent 10 nucleotides cannot be involved in determining the editing site, since they are synthesized only after the relevant RNA editing event is complete. Sequences 5P to an editing site could, on the other hand, interact by backfolding, although this may create steric problems as the RNA polymerase complex covers several nucleotides of the emerging DNA-RNA heteroduplex in the transcription bubble. Certain nucleotide motifs could trigger the insertions, but have so far not been detected in the sequences analyzed to date. It is possible that transacting guide molecules confer speci¢city, but as yet such molecules have not been identi¢ed either in the mitochondrial genome or as imported RNAs encoded in the nucleus. Availability of the complete mitochondrial genome sequence of Physarum may help resolve this question.

7. RNA editing in mitochondria and plastids of plants Mitochondria and plastids of almost all of the land plants examined contain examples of posttranscriptional conversion of C to U and U to C in the sequences of many transcripts [33^45]. In plastids including chloroplasts only between 5 and 20 Cs are found to be deaminated to Us in total (Fig. 6). In mitochondria, by extrapolation from the RNAs analyzed to date, probably up to 500 in some plants up to 1000 C to U changes are expected. In the majority of plant species U to C conversions are very rare events, amounting to only about two or three in the total mitochondrial RNA of any one £owering plant. RNA editing sites are mostly found

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Fig. 6. RNA editing in mitochondria and plastids of plants alters C to U and less frequently U to C. The enzyme(s) responsible deaminate C to U at speci¢c sites in mRNAs and tRNAs. It is as yet unclear how the editing sites are recognized and which enzymes are involved. For the reaction from U to C transaminases and respective cofactors may be involved. Reprinted from [55] with kind permission.

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in coding regions of mRNAs and less frequently in introns and other non-translated regions. In some cases RNA editing in tRNA molecules restores essential base-pairings (Fig. 4). In these instances only complete RNA editing allows correct folding and further maturation and processing of the tRNA precursors. The ribosomal RNAs in plant organelles appear to undergo very little RNA editing. Since no nucleotide insertions or other changes of nucleotide identities have been observed in plants, the most parsimonious explanation of the mechanism of RNA editing would be to propose the existence, and to invoke the activity, of an RNA-speci¢c C-deaminase analogous to the APOBEC-1 enzyme described in the apoB editing of mammalia for example. All the evidence gathered to date does indeed point to a deamination reaction being involved in the C to U conversions. Such deaminases are, however, usually from energetic considerations unlikely to catalyze the reverse reaction of adding an amino group to a U residue to generate a C. How a given C in an RNA molecule is identi¢ed for conversion to a U is at present unclear. Given the sheer numbers of editing sites that need to be speci¢ed in mitochondrial transcripts, a sequence-speci¢c recognition by proteins alone, as in the apoB editing, is extremely unlikely. Current thinking thus favors the coinvolvement of RNA sequences guiding the, as yet hypothetical, editosome to speci¢c sites. Such a guide function may be provided by cis- or trans-acting RNA molecules, but cannot reside in the sequence vicinity of the editing sites alone. No common sequence motifs or related groups of such can be identi¢ed around the di¡erent C to U conversion sites beyond a low percentage of Gs in the nucleotide position preceding the edited Cs. In both mitochondria and plastids downstream nucleotides appear to have little to do with the editing site speci¢cations, while the upstream sequence context is apparently critical. In both types of organelle the essential upstream region varies between di¡erent editing sites, sometimes only 25^30 nucleotides are su¤cient while at other sites 200 nucleotides may not be enough to specify a C to be edited. In mitochondria, sequence duplications have been found, in which RNA editing is correctly maintained as long as su¤cient upstream sequences are present. These di¡er between individual sites as found in experiments with transgenic

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plastids, in which various lengths of upstream and downstream sequence insertions were tested in vivo. RNA editing in plant organelles is a posttranscriptional process as suggested by the identi¢cation of potential RNA editing intermediates. In these partially edited transcripts some Cs have been changed to Us, while at other potential editing sites the Cs encoded by the genome are present. In these partially edited RNA molecules there is no order of editing along the RNA molecule suggesting that the editosome complex does not scan the RNA molecule along its linear length, but rather randomly selects certain sites or regions and releases the transcript again after editing. Partially edited mRNAs are found in polysomal fractions of plant mitochondria and are apparently translated into a family of variant proteins. However, only one type of protein sequence appears to be incorporated into the polypeptide complexes of the respiratory chain. This polypeptide sequence corresponds generally to the polypeptide sequence best conserved with the homologues in other organisms and may be selected by its physiological and biochemical functionality. Indeed it is unlikely that polypeptides synthesized from unedited RNAs would function properly and such proteins would thus impair the e¤ciency of mitochondrial respiration.

8. RNA editing in mitochondria of animals The ¢rst descriptions of RNA editing in mammalian mitochondria were not recognized as such, since they were identi¢ed before the term RNA editing was coined and were more readily explained as being due to the well-established mechanism of polyA addition [46]. Many of the mRNAs in animal mitochondria derive their 3P termini by processing at the 5P ends of downstream tRNAs followed by limited polyadenylation of the mRNA molecules. Some of the nascent mRNA 3P termini do not contain a translational stop codon, but end with for example a U after the last in-frame codon. This U is posttranscriptionally extended to generate a UAA stop codon by 3P addition of a polyA tract. It is not clear whether translation by the mitochondrial ribosome could e¡ectively produce a full-length polypeptide including the last complete codon prior to polyad-

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enylation at the solitary U. Certainly translation would be e¡ectively terminated if the transcript template is incomplete and the ribosome would dissociate. The non-templated completion of the transcript information by polyadenylation may be required for faithful protein biosynthesis or may alternatively be involved in RNA metabolism. Addition of a polyA tract by polyA polymerase is also the underlying mechanism of RNA editing of tRNAs in mitochondria of many metazoan animals [29,47,48]. In the mitochondrial genomes of platypus, chicken and many snails, genes for tRNAs sometimes overlap by one or two nucleotides. The generally correct processing at the respective 5P termini of the tRNAs by RNase P leaves the upstream tRNA lacking the 3P terminal nucleotides. These are added as a short polyA sequence by a polyA polymerase and subsequently trimmed back to generate the mature 3P terminus by 3P processing catalyzed by RNase Z (Fig. 4). RNA editing in animal mitochondria is thus template-independent and utilizes the non-discriminating polyA polymerase, which will accept 3P ends of any RNA molecule as template, be it mRNA, tRNA or rRNA. A template dependence might however be involved in completing the 3P terminus of the tRNA-Ser in the platypus, where the four nucleotides 5P-CCCA-3P have to be added. In postulating that in this instance the polyA polymerase may also catalyze CTP addition to the truncated tRNA, there would be no need for yet another enzymatic activity. However, this simple speculation has yet to be con¢rmed. A di¡erent mechanism of tRNA editing may be operative in marsupial tRNAs, where a C nucleoside in the anticodon of tRNA-Asp is deaminated to a U at least in the American opossum. This deamination is apparently part of the tRNA modi¢cation processes and occurs after 5P and 3P trimming of the initially synthesized tRNA precursor. The enzyme responsible may be a modi¢ed CTP-deaminase or an adapted modi¢cation enzyme the nature of which has not yet been clari¢ed. The biological consequence of this editing is the creation of two distinct tRNA species from just one gene: the unedited tRNA-Asp (GCC) and the edited tRNA-Gly (GUC) are present in about equal proportions in the marsupial mitochondria. The sole di¡erence be-

tween the two tRNAs is this C to U change in the anticodon, which speci¢es the di¡erence in codon recognition as well as altering the speci¢city elements for cognate amino acylation. In this case RNA editing results in two gene products being synthesized from just one coding region and ampli¢es the biological diversity beyond the genomic capacity. This is consistent with the very compact animal mitochon-

Fig. 7. Cotranscriptional RNA editing of RNA virus transcripts inserts non-templated nucleotides by stuttering of the RNA-dependent RNA polymerase. The frame shifts induced by the insertion of one or two nucleotides extend the open reading frames most often of the P gene and the edited mRNA now encodes a much larger protein, usually the V protein. Speci¢c sequence motifs in the viral RNA induce the RNA polymerase complex to stutter and to repeat the last transcribed nucleotide of the template RNA before resuming faithful transcription. Reprinted from [55] with kind permission.

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drial genomes, the size of which appears to be constrained in evolution.

9. Cotranscriptional RNA editing of virus RNAs During transcription of several RNA viruses into mRNA additional nucleotides are incorporated which are not speci¢ed by the viral genome [49^ 51]. In the paramyxoviruses such as measles, Sendai, parain£uenza and mumps viruses one or two and even up to eight or 10 G residues are inserted at speci¢c sites (Fig. 7). In the Ebola viruses additional As are incorporated during transcription, but not replication, as mentioned earlier. All of these RNA viruses, including the paramyxoviruses and the Ebola type, propagate via antisense RNA molecules, which are then transcribed by a virus-encoded RNA-dependent RNA polymerase. This polymerase is prone to pausing and stuttering at certain nucleotide combinations, mostly mono- or oligonucleotide tracts. At the nascent mRNA 3P ends up to several hundred As are added by the same polymerase, although they are not templated. These additional As stabilize the mRNA in a fashion analogous to the polyA tails added by speci¢c polyA polymerases in other systems. While the mRNA polymerase (complex) pauses at these positions, the RNA replicase (complex) faithfully synthesizes the replication intermediate RNA from the virion RNA. Most likely the same RNA polymerase is differentially in£uenced by additional cofactors with replication taking place sheltered in the virion, while transcription to mRNA usually occurs in the cytoplasm. This virus-encoded RNA-dependent RNA polymerase also pauses and incorporates non-templated nucleotides by `stuttering' near the genomic stop codon of the ¢rst open reading frame in the unedited mRNA. Incorporation of one or two Gs or As upstream of the translational stop codon shifts the reading frame, which upon translation generates different proteins with divergent carboxy-terminal sequences. In the mumps and some other viruses the additional amino acid sequence can more than double the size of the genomically predicted ¢rst open reading frame. The pausing and subsequent `stuttering' of the

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RNA polymerase is compared to the sequence-speci¢ed pausing and transcription termination of the E. coli RNA polymerase. Processively unstable transcription complexes are induced in a similar fashion by certain sequences at the template sites. Pyrimidine-rich sequences with long U stretches before several Cs cause the viral RNA polymerase to slow down, slip back one nucleotide on the template and incorporate another G nucleotide opposite the same C again. This stuttering progression alters a sequence of, for example, three Gs predicted by the genomic RNA to four Gs in the edited mRNA. In the RNA viruses this dissociation and realignment of the polymerase-product complex to the previously transcribed nucleotide on the RNA template is explained by the speci¢c rate constants of dissociation and RNA-protein binding. These and the calculated free energy levels are very similar in the di¡erent alignments even when including G-C as well as G-U pairings.

10. RNA editing and its consequences and advantages RNA editing is required for gene expression in many of the systems discussed above. Often the genomic information encoding an open reading frame or a tRNA is cryptic or incomplete and will not yield a functional product. Thus the genetic system involved is dependent on RNA editing for its biological optimization and eventual survival. In the mitochondrial and plastid systems this process is particularly important for the synthesis of functional proteins which after editing exhibit closer sequence conservation with their homologues in other systems. In addition in these organelles RNA editing ensures that tRNAs can fold correctly. Nevertheless, from ¢rst impressions RNA editing is rather extravagant and costly, since without exception all of the RNA editing events described would be rendered unnecessary if the sequence of the mature mRNA was encoded in the genome, as it is in bacteria. We thus have to look for or produce a hypothesis to explain the biological signi¢cance of RNA editing. This could include regulatory control of gene expression at the posttranscriptional level, where the rapid introduction of an AUG translational start codon could rapidly select an RNA for trans-

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lation without requiring the complete de novo synthesis of the transcript. This may be re£ected for example in the observed developmental di¡erences in RNA editing e¤ciency in trypanosomes. In addition, another advantage of RNA editing could be the potential of synthesizing two or more distinct products from a single gene as exempli¢ed by the viral editing systems or in the tissue-speci¢c apoB editing, where two lipid carrier proteins with di¡erent properties are derived from one and the same genomic coding region. Editing in the GluR receptor channels results in increasing the family of variant proteins available for the combinatory assembly of the channels and allows ¢ne-tuning of the chemo-electric potential transmission properties. Here the available variation of a gene family is increased without increasing the number of genes. This presumed advantage may only be illusory as additional polypeptides are required for the editing process. RNA editing thus appears to be rather a biologically sel¢sh process, which has become established and perpetuated.

11. Origin and evolution of RNA editing Considering the diverse types and characteristics of RNA editing (Table 1) and the multitude of di¡erent biochemical and enzymatic processes involved it is obvious that there are several independently established mechanisms which have been grouped under the terminological umbrella of RNA editing. In some types of editing nucleoside conversion is apparently the result of an evolutionary recruitment of modi¢cation enzymes to act on RNA, while in the case of the insertional editings other preexisting enzymes have become assembled into complexes with modi¢ed speci¢cities [6,52]. The animal nuclear RNA editing systems have apparently evolved starting from mononucleotide deaminases, which gave rise to larger gene families including the apobec-1 and adar genes. These exhibit clear evolutionary relationships with the bacterial deaminases involved in nucleotide metabolism. However, the adenosine nucleotide deaminase of E. coli cannot deaminate a nucleoside in the RNA since the reaction pocket in the enzyme is too small to accommodate the RNA strand. This internal active site has

been widened by amino acid changes in the APOBEC-1 and ADAR proteins as modelled in the active site access structures [19,53]. The insertional editing in trypanosome mitochondria has enzymatically nothing in common with the nucleoside conversion processes. The enzymes involved, an endonuclease, terminal U transferase and RNA ligase, have apparently been recruited and adapted from di¡erent sources and contexts totally unrelated to nucleoside modi¢cation. However, the speci¢city determinants of nucleotide insertion in trypanosome mitochondria, as mediated by the interaction of double stranded RNA regions formed of mRNA and guide RNA, are analogous to the tRNA editing processes in animal mitochondria as well as in Acanthamoeba mitochondria and the GluR editing in mammalia. Since all of these RNA editing processes have evolved independently, (partially) basepairing double stranded RNAs in cis or trans can apparently readily be recruited as speci¢city determinants. Such antisense guide sequences may also be involved in the still rather mysterious types of RNA editing such as those in Physarum mitochondria and in plant organelles. Antisense guide RNA molecules also o¡er another connection between RNA editing and modi¢cation, since in eukaryotes ribose methylations of rRNAs are also speci¢ed in trans by guide RNA molecules, the small nucleolar RNAs [54]. The presence of RNA editing in its various guises and appearances suggests that most if not all of these processes have evolved in speci¢c lineages of speciation. None of the diverse RNA editing mechanisms can be convincingly linked to any of the processes assumed to have existed in an ancient primary RNA world. RNA editing mechanisms appear to have evolved much later to compensate for gene sequences gone awry or to increase evolutionary variation.

12. Conclusion and recommendation If you still want to know more about RNA editing, which we hope to have convinced you is a fascinating subject, we recommend a book recently published by the American Society for Microbiology. It is entitled `Modi¢cation and Editing of RNA', edited by Henri Grosjean and Rob Benne [55]. This treatise provides in a collection of essays an up-to-date and

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comprehensive coverage of the di¡erent processes of editing and modi¢cation. The remaining question as to whether RNA editing occurs in bacteria is up to the microbiologist to resolve. Clues that RNA editing may exist could include `sequence errors', unusual amino acid or codon alignments and the like, anything that could be explained by a modi¢cation of a nucleoside or by the addition of a nucleotide in the RNA molecule. One candidate case has been identi¢ed in the tRNA-Ser in E. coli, which exists in two forms containing either a C or a dihydrouridine at nucleotide position 20 [29,56]. Attempts to set up in vitro systems have so far met with mixed success, this event is closely coupled to modi¢cation and/or transcription and may or may not be a genuine case of RNA editing.

[7]

[8]

[9] [10]

[11]

[12] [13] [14]

Acknowledgements

[15]

We gratefully thank C.J. Leaver, Oxford, for his kind and critical comments on the text and for having defused many awkward phrases. Work in the authors' laboratory is supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium fu«r Forschung and the Fonds der Chemischen Industrie.

[16]

[17]

References [18] [1] Benne, R., Van den Burg, J., Brakenho¡, J.P.J., Sloof, P., Van Boom, J.H. and Tromp, M.C. (1986) Major transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides not encoded in the DNA. Cell 46, 819^ 826. [2] Grosjean, H. and Benne, R. (1998) Preface. In: Modi¢cation and Editing of RNA (Grosjean, H. and Benne, R., Eds.). pp. xi^xiii. ASM Press, Washington, DC. [3] Lane, B.G. (1998) Historical perspectives on RNA nucleoside modi¢cations. In: Modi¢cation and Editing of RNA (Grosjean, H. and Benne, R., Eds.), pp. 1^20. ASM Press, Washington, DC. [4] Price, D.H. and Gray, M.W. (1998) Editing of tRNA. In: Modi¢cation and Editing of RNA (Grosjean, H. and Benne, R., Eds.), pp. 289^306. ASM Press, Washington, DC. [5] Benne, R. (1994) RNA editing in trypanosomes. Eur. J. Biochem. 221, 9^23. [6] Arts, G.J. and Benne, R. (1996) Mechanism and evolution of

[19]

[20]

[21]

[22]

315

RNA editing in kinetoplastida. Biochim. Biophys. Acta 1307, 39^54. Alfonzo, J.D., Thiemann, T. and Simpson, L. (1997) The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria. Nucleic Acids Res. 25, 3751^3759. Mahendran, R., Spottswood, M.R. and Miller, D.L. (1991) RNA editing by cytidine insertion in mitochondria of Physarum polycephalum. Nature 349, 434^438. Cattaneo, R. (1991) Di¡erent types of messenger RNA editing. Annu. Rev. Genet. 25, 71^88. Auxilien, S. and Grosjean, H. (1995) Edition des ARN d'eucaryotes et viraux par de¨samination enzymatique d'ade¨nosines en inosines. Me¨decine/Sciences 11, 1089^1098. Chang, B.H.-J., Lau, P.P. and Chan, L. (1998) Apolipoprotein B mRNA editing. In: Modi¢cation and Editing of RNA (Grosjean, H. and Benne, R., Eds.), pp. 325^342. ASM Press, Washington, DC. Scott, J. (1995) A place in the world for RNA editing. Cell 81, 833^836. Scott, J. (1997) Message change for a fat controller. Nature 387, 242^243. Smith, H.C. and Sowden, M.P. (1996) Base-modi¢cation mRNA editing through deamination ^ the good, the bad and the unregulated. Trends Genet. 12, 418^424. Chen, S.H., Habib, G., Yang, C.Y., Gu, Z.W., Lee, B.R., Weng, S.A., Silberman, S.R., Cai, S.J., Deslypere, J.P., Rosseneu, M., Gotto Jr., A.M., Li, W.H. and Chan, L. (1987) Apolipoprotein B-48 is the product of a messenger RNA with an organ-speci¢c in-frame stop codon. Science 238, 363^366. Greeve, J., Navaratnam, N. and Scott, J. (1991) Characterization of the apolipoprotein B mRNA editing enzyme : no similarity to the proposed mechanism of RNA editing in kinetoplastid protozoa. Nucleic Acids Res. 19, 3569^3576. Harris, S.G., Sabio, I., Mayer, E., Steinberg, M.F., Backus, J.W., Sparks, J.D., Sparks, C.E. and Smith, H.C. (1993) Extract-speci¢c heterogeneity in high-order complexes containing apolipoprotein B mRNA editing activity and RNA-binding proteins. J. Biol. Chem. 268, 7382^7392. Powell, L.M., Wallis, S.C., Pease, R.J., Ewards, Y.H., Knott, T.J. and Scott, J. (1987) A novel form of tissue-speci¢c RNA processing produces apolipoprotein-B48 in intestine. Cell 50, 831^840. Carter, C.W. (1998) Nucleoside deaminases for cytidine and adenosine: comparisons with deaminases acting on RNA. In: Modi¢cation and Editing of RNA (Grosjean, H. and Benne, R., Eds.), pp. 363^376. ASM Press, Washington, DC. Higuchi, M., Single, F.N., Ko«hler, M., Sommer, B., Sprengel, R. and Seeburg, P.H. (1993) RNA editing of AMPA receptor subunit GluR-B : A base-paired intron-exon structure determines position and e¤ciency. Cell 75, 1361^1370. Hough, R.F. and Bass, B.L. (1997) Analysis of Xenopus dsRNA deaminase cDNAs reveals similarities to DNA methyltransferases. RNA 3, 356^370. Blum, B., Bakalara, N. and Simpson, L. (1990) A model for RNA editing in kinetoplastid mitochondria : `Guide' RNA molecules transcribed from maxicircle DNA provide the edited information. Cell 60, 189^198.

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A. Brennicke et al. / FEMS Microbiology Reviews 23 (1999) 297^316

[23] Kable, M.L., Heidmann, S. and Stuart, K.D. (1997) RNA editing : getting U into RNA. Trends Biochem. Sci. 22, 162^ 166. [24] Simpson, L. and Thiemann, O.H. (1995) Sense from nonsense : RNA editing in mitochondria of kinetoplastid protozoa and slime molds. Cell 81, 837^840. [25] Stuart, K. (1991) RNA editing in mitochondrial mRNA of trypanosomatids. Trends Biochem. Sci. 16, 68^72. [26] van der Spek, H., Arts, G.J., Zwaal, R.R., van den Burg, J., Sloof, P. and Benne, R. (1991) Conserved genes encode guide RNAs in mitochondria of Crithidia fasciculata. EMBO J. 10, 1217^1224. [27] Hajduk, S.L. and Sabatini, R.S. (1998) Mitochondrial mRNA editing in kinetoplastid protozoa. In: Modi¢cation and Editing of RNA (Grosjean, H. and Benne, R., Eds.), pp. 377^394. ASM Press, Washington, DC. [28] Lonergan, K.M. and Gray, M.W. (1993) Editing of transfer RNAs in Acanthamoeba castellanii mitochondria. Science 259, 812^816. [29] Lonergan, K.M. and Gray, M.W. (1993) Predicted editing of additional transfer RNAs in Acanthamoeba castellanii mitochondria. Nucleic Acids Res. 21, 4402. [30] Laforest, M.J., Roewer, I. and Lang, B.F. (1997) Mitochondrial tRNAs in the lower fungus Spizellomyces punctatus : tRNA editing and UAG `stop' codons recognized as leucine. Nucleic Acids Res. 25, 626^632. [31] Mahendran, R., Spottswood, M.S., Ghate, A., Ling, M.L., Jeng, K. and Miller, D.L. (1994) Editing of the mitochondrial small subunit rRNA in Physarum polycephalum. EMBO J. 13, 232^240. [32] Gott, J.M. and Visomirski-Robic, L.M. (1998) RNA editing in Physarum mitochondria. In: Modi¢cation and Editing of RNA (Grosjean, H. and Benne, R., Eds.), pp. 395^412. ASM Press, Washington, DC. [33] Covello, P.S. and Gray, M.W. (1989) RNA editing in plant mitochondria. Nature 341, 662^666. [34] Gualberto, J.M., Lamattina, L., Bonnard, G., Weil, J.H. and Grienenberger, J.M. (1989) RNA editing in wheat mitochondria results in the conservation of protein sequences. Nature 341, 660^662. [35] Hiesel, R., Wissinger, B., Schuster, W. and Brennicke, A. (1989) RNA editing in plant mitochondria. Science 246, 1632^1634. [36] Hoch, B., Maier, R.M., Appel, K., Igloi, G.L. and Ko«ssel, H. (1991) Editing of a chloroplast mRNA by creation of an initiation codon. Nature 353, 178^180. [37] Pring, D., Brennicke, A. and Schuster, W. (1993) RNA editing gives a new meaning to the genetic information in mitochondria and chloroplasts. Plant Mol. Biol. 21, 1163^1170. [38] Wissinger, B., Brennicke, A. and Schuster, W. (1992) Regeneration good sense : RNA editing and trans splicing in plant mitochondria. Trends Genet. 8, 322^328. [39] Grienenberger, J.M. (1993) RNA editing in plant organelles. In : RNA Editing (Benne, R., Ed.), Ellis Harwood, New York. [40] Malek, O., La«ttig, K., Hiesel, R., Brennicke, A. and Knoop, V. (1996) RNA editing in bryophytes and a molecular phylogeny of land plants. EMBO J. 15, 1403^1411.

[41] Freyer, R., Kiefer-Meyer, M.C. and Ko«ssel, H. (1997) Occurrence of plastid RNA editing in all major lineages of land plants. Proc. Natl. Acad. Sci. USA 12, 6285^6296. [42] Dietrich, A., Small, I., Cosset, A., Weil, J.H. and Mare¨chalDrouard, L. (1996) Editing and import: Strategies for providing plant mitochondria with a complete set of functional transfer RNAs. Biochimie 75, 518^529. [43] Bock, R., Hermann, M. and Fuchs, M. (1997) Identi¢cation of critical nucleotide positions for plastid RNA editing site recognition. RNA 3, 1194^1299. [44] Gray, M.W. and Covello, P.S. (1993) RNA editing in plant mitochondria and chloroplasts. FASEB J. 7, 64^71. [45] Marchfelder, A., Binder, S., Brennicke, A. and Knoop, V. (1998) Preface. In : Modi¢cation and Editing of RNA (Grosjean, H. and Benne, R., Eds.), pp. 307^323. ASM Press, Washington, DC. [46] Clayton, D.A. (1992) Transcription and replication of animal mitochondrial DNAs. Int. Rev. Cytol. 141, 217^232. [47] Bo«rner, G.V., Mo«rl, M., Janke, A. and Pa«a«bo, S. (1996) RNA editing changes the identity of a mitochondrial tRNA in marsupials. EMBO J. 15, 5949^5957. [48] Yokobori, S.I. and Pa«a«bo, S. (1995) Transfer RNA editing in land snail mitochondria. Proc. Natl. Acad. Sci. USA 92, 10432^10435. [49] Curran, J., Boeck, R. and Kolakofsky, D. (1991) The Sendai virus P gene expresses both an essential protein and an inhibitor of RNA synthesis by shu¥ing modules via mRNA editing. EMBO J. 10, 3079^3085. [50] Zheng, H., Fu, T.B., Lazinski, D. and Taylor, J. (1992) Editing on the genomic RNA of human hepatitis delta virus. J. Virol. 66, 4693^4697. [51] Kolakofsky, D. and Hausmann, S. (1998) Cotranscriptional paramyxovirus mRNA editing: a contradiction in terms? In: Modi¢cation and Editing of RNA (Grosjean, H. and Benne, R., Eds.), pp. 413^420. ASM Press, Washington, DC. [52] Covello, P.S. and Gray, M.W. (1993) On the evolution of RNA editing. Trends Genet. 9, 265^268. [53] Navaratnam, N., Fujino, T., Bayliss, J., Jarmuz, A., How, A., Richardson, N., Somasekaram, A., Bhattacharya, S., Carter, C. and Scott, J. (1998) Escherichia coli cytidine deaminase provides a molecular model for ApoB RNA editing and a mechanism for RNA substrate recognition. J. Mol. Biol. 275, 695^714. [54] Bachellerie, J.-P. and Cavaille, J. (1998) Small nucleolar RNAs guide the ribose methylations of eukaryotic rRNAs. In: Modi¢cation and Editing of RNA (Grosjean, H. and Benne, R., Eds.), pp. 255^272. ASM Press, Washington, DC. [55] Grosjean, H. and Benne, R. (1998) Modi¢cation and Editing of RNA. ASM Press, Washington, DC. [56] Grosjean, H., Nicoghosian, E., Haumont, E., So«ll, D. and Cedergren, R. (1985) Nucleotide sequences of two serine tRNAs with a GGA anticodon: the structure-function relationships in the serine family of E. coli tRNAs. Nucleic Acids Res. 13, 5697^5706.

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