TIBS 1 7 - JANUARY 1992
RNA editing: in In the past few years, several cases have been described in which the coding potential of a primary transcript is altered by mechanisms other than RNA splicing. These quite disparate instances of RNA modification have been collectively termed messenger RNA editing, a term which suggests the correction of apparent coding errors 0.e. frameshifts or 'wrong' residues), although the modifications can involve insertion and deletion of hundreds of residues, sometimes even creating new reading frames ~,2. Since, of the six instances of RNA editing described 3, four concern mitochondrial systems, another a viral system and only one a nuclear gene, RNA editing could have been considered an interesting phenomenon of limited relevance. However, two newly described cases of editing, one concerning at least two transcripts of chloroplasts 4, and the other at least three nuclear transcripts in the mammalian brain 5, further broaden the impact of RNA editing and suggest that more unrecognized editing cases exist.
chloroplast and U br, in Types and mechanisms of mRNA editing
Four of the first six types of RNA editing described are based on the insertion of residues (Fig. 1). (1) In several mitochondrial transcripts of trypanosomes, few or hundreds of uridine (U) residues are inserted or deleted L2. This is probably achieved by transesterification, instructed and carried out by short 'guide' RNAsE (2) In a mitochondrial transcript of a slime mold, single cytidine (C) residues are added at multiple positions 7 by an unknown mechanism. (3) Cotranscriptional polymerase stuttering results in the insertion of guanosine (G) residues in a transcript of the paramyxovirus class of RNA viruses that includes measles and mumps (see Ref. 3 for review). (4) In transcripts of vertebrate mitochondria, UAA and UGA stop codons are reconstituted by polyTranscripts of Residues i n s e r t e d / m o d i f i e d adenylation3; a similar phenomenon was ob(1) mitochondria of ...AAUUUAUGUUGUcUuu... trypanosomes served in a nuclear transcript 8. (2) mitochondria of ...AuCucUAAGGGUUUAACCGG... P. p o l y c e p h a l u m i = The two recently identFRAMESHIFT FRAMESHIF-T ified types of editing, and two previously described (3) paramyxoviruses ...AUUA.8.~,AAGGGGCACAC... (P gene) i FRAMESHIFT forms, involve the conversion of residues. (5) In (4) mitochondria of ...CAGUAAAAAAAAAAA.. the mammalian apolipovertebrates ~ ' STOP protein B mRNA, a nuclear transcript, a C is converted to a U by (5) apolipoprotein B ...GAUAUAAUUUGAUCAGUAUA... deamination. This causes gene of mammals STOP the tissue-specific generation of a stop codon (6) mitochondria of ...uuuUucAUUGUGGUUUAC... (see Ref. 9 for review). (6) higher plants i = , , PHE TYR In many, if not all, mitochondrial transcripts of (7) chloroplasts of ...AAUAAUAUGGCGAAACAU... higher plants i i several species of higher START plants C to U and, to a (8) glutamate-gated ion ...UUUAUGCGGCAAGGA... lesser extent, U to C conchannels of mammals ~ versions are found (see ARG Ref. 10 for review). This form of mRNA editing, Figure 1whose mechanism is still Different types of messenger RNA editing. Bold characunknown, might be the ters represent residues inserted (1-4) or modified one concerning the (5-8). The bold G residue in the glutamate-gated ion largest number of organchannel mRNA sequence (8) might in fact be an inosine (I). See text for details and references. isms. The two recently
identified forms of RNA editing (Fig. 1, 7 and 8) are described in some detail below.
RNA editing in the chloroplast RNA editing by C to U conversion has also been shown to occur in chloroplast transcripts of two different plant species, maize and tobacco 4. Remarkably, the predicted protein products of some chloroplast genes of certain plant species contain variant amino acids at positions completely conserved among other species. For example, in the genomic sequences of the rpl2 gene of the maize chloroplast and of the psbL gene of the tobacco chloroplast, an ACG codon appears at the position where the initiation codon ATG would be expected. This observation prompted Hans KBssel's group to compare the genomic sequences with cDNA sequences of the corresponding mRNAs. Indeed, in the rpl2 maize transcript a C to U conversion which creates the predicted AUG initiation codon was inferred; the same was true for the psbL tobacco transcript. No additional C to U conversions were inferred in the approximately 450 nucleotides analysed in the rpl2 transcript, indicating that RNA editing in chloroplasts might be less extensive than in plant mitochondria. As mentioned above, C to U conversions occur both in nuclear and in mitochondrial transcripts. Different mechanisms, however, underlie nuclear and mitochondrial C to U conversions. In the case of apolipoprotein B, it was demonstrated experimentally that conversion is largely determined by conserved sequences flanking the editing siteg; in mitochondrial transcripts the sequences flanking different editing sites are completely divergent 1°. Moreover, a cytoplasmic S100 in vitro editing system cannot convert sites which are edited in mitochondria n. It will be interesting to see if sequence or structural determinants are more important in chloroplast RNA editing, and if in this system C to U conversion is achieved by deamination or by other mechanisms such as nucleotide substitution or transglycosylation9.
RNA editing in the brain Another illuminating observation was recently made by studying genes and cDNAs of transcripts coding for ion channels mediating fast excitatory
© 1992, Elsevier Science Publishers, (UK)
TIBS 17 - J A N U A R Y 1 9 9 2
synaptic transmission in the mammalian central nervous system. It was previously known that subunit components of two related classes of glutamate receptor channels harbor either a glutamine or an arginine in a defined position of their putative channelforming segment. The arginine residue in this segment profoundly alters the properties of ion flow. Peter Seeburg, Rolf Sprengel and colleagues have now demonstrated 5 that the genomic DNA sequences encoding the homologous segment of all six subunits studied harbor a glutamine codon (CAG), even though an arginine codon (CGG) is found in cDNAs made from mRNAs of three subunits. Interestingly, the transcripts of one subunit of one sequence class (called here class I) are edited with 100% efficiency, whereas the transcripts of the other three subunits of this class are not edited, in spite of 90-95% homology over 30 nucleotides surrounding the editing site. The transcripts of the two available subunits of the other class are edited with 40% and 80% efficiency, respectively. Sommer et alp thus speculate that two editing complexes with slightly different properties account for editing of the two classes of transcripts. To explain the selectivity of editing in the case of class I transcripts, it can be postulated that distal sequences which differ among subunits are the principal editing determinant. Sommer et al. also speculate that the apparent A to G conversions detected in cDNAs of brain transcripts could be a consequence of the reverse transcription process, and that the editing events could in fact be deaminations of As to inosines. Inosines will base pair with Cs, rather than with Us, during reverse transcription. Synthesis of the second strand will result in A to G substitutions in the cDNAs. The only adenosine deaminase known to act on eukaryotic mRNAs is an RNA unwindingmodifying activity which is dependent on double-stranded RNA12J3. It was recently demonstrated that this activity can use as a substrate not only long intermolecular RNA hybrids but also short (20-25) intramolecular hybrids ~4,~s. It is conceivable that an intramolecular hybrid could form between the region surrounding the editing site and a complementary region situated in the intron located immediately downstream of the editing site. The existence of a short region of homology in only one of the four primary transcripts of the same
class would explain the strict editing selectivity. Alternatively, a secondary structure-specific editing complex could be postulated, since secondary structure- and sequence-specific enzymes modify tRNAs 16J7. Perspectives
RNA editing is not limited to mitochondrial transcripts; different editing types are also found in nuclear, chloroplast and viral transcripts. RNA editing is yet another pitfall in the prediction of protein sequences starting from genes. It joins other sources of confusion such as slightly modified genetic codes, genomic rearrangements, RNA splicing, complications at initiation and termination of translation, and ribosomal frameshifting. RNA editing adds an additional level in the control of gene expression; other central effects of RNA editing on important biological functions may well be disclosed in future. The existence of many different RNA editing mechanisms indicates that the information content of messenger RNA is more flexible than previously thought, although how manipulation of genetic information is controlled remains to be determined. Finally, certain RNA editing mechanisms might be direct descendants of mechanisms used in the replication of primitive genomes; RNA editing might thus open a window on the elusive primordial RNA world.
Acknowledgements
Thanks are due to David Stern for critical reading of the manuscript. R. C. is a START fellow of the Schweizerische Nationalfonds. References 1 Benne,R. et al. (1986) Cell 46, 819-826 2 Stuart, K. (1991) Trends Biochem. Sci. 16, 68-72 3 Cattaneo,R. (1991) Annu. Rev. Genet. 25, 71-88 4 Hoch, B. et al. (1991) Nature 353, 178-180 5 Sommer, B. et al. (1991) Cell 67, 11-19 6 Blum, B. et al. (1991) Cell 65, 543-550 7 Mahendran,R., Spottswood,M. R. and Miller, D. L. (1991) Nature 349, 434-438 8 Jenh, C-H. et al. (1986) Prec. Natl Acad. Sci. USA 83, 8482-8486 9 Scott, J. (1992) Trends Biechem. Sci. 17, (Februaryissue) 10 Walbot, V. (1991) Trends Genet. 7, 37-39 11 Navaratnam,N. et al. (1991) Nucleic Acids Res. 19, 1741-1744 12 Bass, B. L. and Weintraub, H. (1988) Cell 55, 1089-1098 13 Wagner, R. W. et al. (1989) Prec. Natl Acad. Sci. USA 86, 2647-2651 14 Sharmeen,L. et al. (1991) Prec. Natl Acad. Sci. USA 88, 8096-8100 15 Nishikura, K. et al. (1991) EMBO J. 10, 3523-3532 16 Elliott, M. S. and Trewyn, R. W. (1984) J. Biol. Chem. 259, 2407-2410 17 Bj6rk, G. R. et al. (1987)Annu. Rev. Biochem. 56, 263-287
ROBERTO C A T r A N E O Departments of Pathology and Cell Biology, Yale University School of Medicine, PO Box 3333, New Haven, CT 06510-8023, USA.
of the m~or advantages of membership of the UK B i ~ m c a l S o c ~ ~ ~,,=ys ~ that the Society does not make any charge to ~ attending it.s m ~ ~ i e ~ c , ~ of ~ h are held per year attracting M r 1000 part . meet~ ~ over 3-5 days, often @ up to five s ~ t a n e o u s colloquia. A daily registration fee is n o ~ l y c h a ~ to n ~ r s . ~ Society is, ~ r , pleased to announce that it ~ ' n o w w a ~ the d ~ atte~ance fee for any scientists n o are m ~ of a national ~iety ~ affiliated to the International ~ of Biochemistry and ~ u l a r ~ o r the ~ i o n of E u r O ~ Biochemical Societies. All are, of c o ~ , welcor~ to join .as f u l l ~ the Society, which ~ a ~ n to ~ a t t e ~ c e at all meetings also brings Other raembership ser. vices; e. g. the ~ The Biochemist ~ ~ s s to travel grants. The Biochemi~ Soci~has also revkrwedother ~ s of its m e ~ l p and ~ a~ in particular those w h ~ are avai~te to students and younger ~ s t s . Of the @ g mem~ berstdp of over 8000 there ~ some 1800 overseas members. However, up until now ~ship (currer~ £10 per year) has been limited to those residing in ~ United
The special student membership rate will apply from registration for first degree to two years after graduation as PhD, normally up to a maximum of six years in all. The Biochemical Society has also introduced a new travel grant system specially for ~ n t s . From 1 January 1992, all student members can apply once a year for a grant to attend meetings sponsored by the Society. The grant will cover one night's accommodation and/or travel up tea maximum of £50. Foreign student members may apply for this grant in c o n ~ with their travel and accommodation in the UK.
5
RNA editing: in In the past few years, several cases have been described in which the coding potential of a primary transcript is altered by mechanisms other than RNA splicing. These quite disparate instances of RNA modification have been collectively termed messenger RNA editing, a term which suggests the correction of apparent coding errors 0.e. frameshifts or 'wrong' residues), although the modifications can involve insertion and deletion of hundreds of residues, sometimes even creating new reading frames ~,2. Since, of the six instances of RNA editing described 3, four concern mitochondrial systems, another a viral system and only one a nuclear gene, RNA editing could have been considered an interesting phenomenon of limited relevance. However, two newly described cases of editing, one concerning at least two transcripts of chloroplasts 4, and the other at least three nuclear transcripts in the mammalian brain 5, further broaden the impact of RNA editing and suggest that more unrecognized editing cases exist.
chloroplast and U br, in Types and mechanisms of mRNA editing
Four of the first six types of RNA editing described are based on the insertion of residues (Fig. 1). (1) In several mitochondrial transcripts of trypanosomes, few or hundreds of uridine (U) residues are inserted or deleted L2. This is probably achieved by transesterification, instructed and carried out by short 'guide' RNAsE (2) In a mitochondrial transcript of a slime mold, single cytidine (C) residues are added at multiple positions 7 by an unknown mechanism. (3) Cotranscriptional polymerase stuttering results in the insertion of guanosine (G) residues in a transcript of the paramyxovirus class of RNA viruses that includes measles and mumps (see Ref. 3 for review). (4) In transcripts of vertebrate mitochondria, UAA and UGA stop codons are reconstituted by polyTranscripts of Residues i n s e r t e d / m o d i f i e d adenylation3; a similar phenomenon was ob(1) mitochondria of ...AAUUUAUGUUGUcUuu... trypanosomes served in a nuclear transcript 8. (2) mitochondria of ...AuCucUAAGGGUUUAACCGG... P. p o l y c e p h a l u m i = The two recently identFRAMESHIFT FRAMESHIF-T ified types of editing, and two previously described (3) paramyxoviruses ...AUUA.8.~,AAGGGGCACAC... (P gene) i FRAMESHIFT forms, involve the conversion of residues. (5) In (4) mitochondria of ...CAGUAAAAAAAAAAA.. the mammalian apolipovertebrates ~ ' STOP protein B mRNA, a nuclear transcript, a C is converted to a U by (5) apolipoprotein B ...GAUAUAAUUUGAUCAGUAUA... deamination. This causes gene of mammals STOP the tissue-specific generation of a stop codon (6) mitochondria of ...uuuUucAUUGUGGUUUAC... (see Ref. 9 for review). (6) higher plants i = , , PHE TYR In many, if not all, mitochondrial transcripts of (7) chloroplasts of ...AAUAAUAUGGCGAAACAU... higher plants i i several species of higher START plants C to U and, to a (8) glutamate-gated ion ...UUUAUGCGGCAAGGA... lesser extent, U to C conchannels of mammals ~ versions are found (see ARG Ref. 10 for review). This form of mRNA editing, Figure 1whose mechanism is still Different types of messenger RNA editing. Bold characunknown, might be the ters represent residues inserted (1-4) or modified one concerning the (5-8). The bold G residue in the glutamate-gated ion largest number of organchannel mRNA sequence (8) might in fact be an inosine (I). See text for details and references. isms. The two recently
identified forms of RNA editing (Fig. 1, 7 and 8) are described in some detail below.
RNA editing in the chloroplast RNA editing by C to U conversion has also been shown to occur in chloroplast transcripts of two different plant species, maize and tobacco 4. Remarkably, the predicted protein products of some chloroplast genes of certain plant species contain variant amino acids at positions completely conserved among other species. For example, in the genomic sequences of the rpl2 gene of the maize chloroplast and of the psbL gene of the tobacco chloroplast, an ACG codon appears at the position where the initiation codon ATG would be expected. This observation prompted Hans KBssel's group to compare the genomic sequences with cDNA sequences of the corresponding mRNAs. Indeed, in the rpl2 maize transcript a C to U conversion which creates the predicted AUG initiation codon was inferred; the same was true for the psbL tobacco transcript. No additional C to U conversions were inferred in the approximately 450 nucleotides analysed in the rpl2 transcript, indicating that RNA editing in chloroplasts might be less extensive than in plant mitochondria. As mentioned above, C to U conversions occur both in nuclear and in mitochondrial transcripts. Different mechanisms, however, underlie nuclear and mitochondrial C to U conversions. In the case of apolipoprotein B, it was demonstrated experimentally that conversion is largely determined by conserved sequences flanking the editing siteg; in mitochondrial transcripts the sequences flanking different editing sites are completely divergent 1°. Moreover, a cytoplasmic S100 in vitro editing system cannot convert sites which are edited in mitochondria n. It will be interesting to see if sequence or structural determinants are more important in chloroplast RNA editing, and if in this system C to U conversion is achieved by deamination or by other mechanisms such as nucleotide substitution or transglycosylation9.
RNA editing in the brain Another illuminating observation was recently made by studying genes and cDNAs of transcripts coding for ion channels mediating fast excitatory
© 1992, Elsevier Science Publishers, (UK)
TIBS 17 - J A N U A R Y 1 9 9 2
synaptic transmission in the mammalian central nervous system. It was previously known that subunit components of two related classes of glutamate receptor channels harbor either a glutamine or an arginine in a defined position of their putative channelforming segment. The arginine residue in this segment profoundly alters the properties of ion flow. Peter Seeburg, Rolf Sprengel and colleagues have now demonstrated 5 that the genomic DNA sequences encoding the homologous segment of all six subunits studied harbor a glutamine codon (CAG), even though an arginine codon (CGG) is found in cDNAs made from mRNAs of three subunits. Interestingly, the transcripts of one subunit of one sequence class (called here class I) are edited with 100% efficiency, whereas the transcripts of the other three subunits of this class are not edited, in spite of 90-95% homology over 30 nucleotides surrounding the editing site. The transcripts of the two available subunits of the other class are edited with 40% and 80% efficiency, respectively. Sommer et alp thus speculate that two editing complexes with slightly different properties account for editing of the two classes of transcripts. To explain the selectivity of editing in the case of class I transcripts, it can be postulated that distal sequences which differ among subunits are the principal editing determinant. Sommer et al. also speculate that the apparent A to G conversions detected in cDNAs of brain transcripts could be a consequence of the reverse transcription process, and that the editing events could in fact be deaminations of As to inosines. Inosines will base pair with Cs, rather than with Us, during reverse transcription. Synthesis of the second strand will result in A to G substitutions in the cDNAs. The only adenosine deaminase known to act on eukaryotic mRNAs is an RNA unwindingmodifying activity which is dependent on double-stranded RNA12J3. It was recently demonstrated that this activity can use as a substrate not only long intermolecular RNA hybrids but also short (20-25) intramolecular hybrids ~4,~s. It is conceivable that an intramolecular hybrid could form between the region surrounding the editing site and a complementary region situated in the intron located immediately downstream of the editing site. The existence of a short region of homology in only one of the four primary transcripts of the same
class would explain the strict editing selectivity. Alternatively, a secondary structure-specific editing complex could be postulated, since secondary structure- and sequence-specific enzymes modify tRNAs 16J7. Perspectives
RNA editing is not limited to mitochondrial transcripts; different editing types are also found in nuclear, chloroplast and viral transcripts. RNA editing is yet another pitfall in the prediction of protein sequences starting from genes. It joins other sources of confusion such as slightly modified genetic codes, genomic rearrangements, RNA splicing, complications at initiation and termination of translation, and ribosomal frameshifting. RNA editing adds an additional level in the control of gene expression; other central effects of RNA editing on important biological functions may well be disclosed in future. The existence of many different RNA editing mechanisms indicates that the information content of messenger RNA is more flexible than previously thought, although how manipulation of genetic information is controlled remains to be determined. Finally, certain RNA editing mechanisms might be direct descendants of mechanisms used in the replication of primitive genomes; RNA editing might thus open a window on the elusive primordial RNA world.
Acknowledgements
Thanks are due to David Stern for critical reading of the manuscript. R. C. is a START fellow of the Schweizerische Nationalfonds. References 1 Benne,R. et al. (1986) Cell 46, 819-826 2 Stuart, K. (1991) Trends Biochem. Sci. 16, 68-72 3 Cattaneo,R. (1991) Annu. Rev. Genet. 25, 71-88 4 Hoch, B. et al. (1991) Nature 353, 178-180 5 Sommer, B. et al. (1991) Cell 67, 11-19 6 Blum, B. et al. (1991) Cell 65, 543-550 7 Mahendran,R., Spottswood,M. R. and Miller, D. L. (1991) Nature 349, 434-438 8 Jenh, C-H. et al. (1986) Prec. Natl Acad. Sci. USA 83, 8482-8486 9 Scott, J. (1992) Trends Biechem. Sci. 17, (Februaryissue) 10 Walbot, V. (1991) Trends Genet. 7, 37-39 11 Navaratnam,N. et al. (1991) Nucleic Acids Res. 19, 1741-1744 12 Bass, B. L. and Weintraub, H. (1988) Cell 55, 1089-1098 13 Wagner, R. W. et al. (1989) Prec. Natl Acad. Sci. USA 86, 2647-2651 14 Sharmeen,L. et al. (1991) Prec. Natl Acad. Sci. USA 88, 8096-8100 15 Nishikura, K. et al. (1991) EMBO J. 10, 3523-3532 16 Elliott, M. S. and Trewyn, R. W. (1984) J. Biol. Chem. 259, 2407-2410 17 Bj6rk, G. R. et al. (1987)Annu. Rev. Biochem. 56, 263-287
ROBERTO C A T r A N E O Departments of Pathology and Cell Biology, Yale University School of Medicine, PO Box 3333, New Haven, CT 06510-8023, USA.
of the m~or advantages of membership of the UK B i ~ m c a l S o c ~ ~ ~,,=ys ~ that the Society does not make any charge to ~ attending it.s m ~ ~ i e ~ c , ~ of ~ h are held per year attracting M r 1000 part . meet~ ~ over 3-5 days, often @ up to five s ~ t a n e o u s colloquia. A daily registration fee is n o ~ l y c h a ~ to n ~ r s . ~ Society is, ~ r , pleased to announce that it ~ ' n o w w a ~ the d ~ atte~ance fee for any scientists n o are m ~ of a national ~iety ~ affiliated to the International ~ of Biochemistry and ~ u l a r ~ o r the ~ i o n of E u r O ~ Biochemical Societies. All are, of c o ~ , welcor~ to join .as f u l l ~ the Society, which ~ a ~ n to ~ a t t e ~ c e at all meetings also brings Other raembership ser. vices; e. g. the ~ The Biochemist ~ ~ s s to travel grants. The Biochemi~ Soci~has also revkrwedother ~ s of its m e ~ l p and ~ a~ in particular those w h ~ are avai~te to students and younger ~ s t s . Of the @ g mem~ berstdp of over 8000 there ~ some 1800 overseas members. However, up until now ~ship (currer~ £10 per year) has been limited to those residing in ~ United
The special student membership rate will apply from registration for first degree to two years after graduation as PhD, normally up to a maximum of six years in all. The Biochemical Society has also introduced a new travel grant system specially for ~ n t s . From 1 January 1992, all student members can apply once a year for a grant to attend meetings sponsored by the Society. The grant will cover one night's accommodation and/or travel up tea maximum of £50. Foreign student members may apply for this grant in c o n ~ with their travel and accommodation in the UK.
5
