MOLECULAR AND CELLULAR BIOLOGY, Aug. 1991, p. 4278-4281 0270-7306/91/084278-04$02.00/0 Copyright C 1991, American Society for Microbiology
Vol. 11, No. 8
RNA Editing Intermediates of cox2 Transcripts in Maize Mitochondria AUSTIN J. YANG AND R. MICHAEL MULLIGAN* Department of Developmental and Cell Biology, University of California, Irvine, California 92717 Received 26 February 1991/Accepted 1 May 1991
Eighteen cytidines are changed to uridines in the coding sequence of transcripts for cytochrome c oxidase subunit 2 (cox2) in maize mitochondria. The temporal relationship of editing and splicing was examined in cox2 transcripts by sequence analysis of spliced and unspliced cDNAs. Cloned cDNAs of unspliced cox2 transcripts ranged from clones with no edited nucleotides to completely edited forms, while spliced cDNAs were nearly completely edited. Incompletely edited transcripts in the nascent pool of unspliced transcripts represent intermediates of the editing process. These results indicate that editing proceeds without a strong directional bias and suggest that RNA editing is a posttranscriptional process.
RNA editing has been reported in the mitochondria of several organisms (1, 5, 6), including higher plant species (3, 9, 11). The nucleotide modifications observed in plant mitochondrial transcripts are usually C-to-U transitions and occasionally U-to-C changes (3, 9-11, 16). RNA editing occurs frequently in the coding sequence and usually results in a radical change of the amino acid specified by the codon
UGAADUUGGUGAGuACUAUwUCCADDCADAAAUCAUCCDU
that restores the evolutionarily conserved sequence of the polypeptide (3, 9, 11). The RNA sequences of cox2 transcripts were determined by direct RNA sequence analysis from maize polysomal RNA (Fig. 1) and is in accord with RNA sequence analysis recently reported for maize mitochondrial cox2 transcripts (4). Total polysomal RNA was obtained from 7-day-old
UCDACUCACDCUCGGDUGGUCCUACDUCUGGUGCUGCCA
2 I I3E S FILT I A L C D A A K P N Q L I L R H L C R M G AUG ADD CUD CGU UCA UUA GAA UGU CGA UOC CUC ACA AUC GCU CUU UGU GAU GCU GCG GAA CCA DOG CAA UDA GGA WA UU 4 S
0
D
A
A
T
Is I I D L D I F F F L I H H L
K
0
G
I
E
I
I
R
T
I
F
S
N
D
G
V
L
V
D
Y
S
D
Y
N
S
S
D
E
0
N
A
N
P
OcU CAA GAC GCA GCA ACA CCu AUG AUG CAA GGA AuC Auu GAC UuA CAu CAC GAD AUG DD uDc Uc COC ADD CDG 5 C L V K L V R A L N H F N K 0 T N P I P Q I F V S R ADD DDG GDD UUC GUA ucA CGG AUG UUG GDD CGC GCU DDA UGG CAu UGc AAC GAG CAA Acu AAu CCA AUc CCG CAA
uwL
IDAOGG
R
I
V
H
S
F
A
W
Y
G
L
N
T
T
7
100 P
S
V
I
P
L
F
I
A
I
I
T
I
K
A
I
G
H
S
L
T
F
D
S
Y
V
P
AGG ADD GDD CAD GGA Acu Acu AUc GAA ADD ADu CGG ACC Auu DDw CCu AGU GUC ADD CCA UUG DUC AUD GCU AuA P
CCA
UCG DUD GCU 9
Q CAA
I
ADD T
R
L L Y CUG DDA uAc SPLICZ SITf T
UGOG UA
Y
E
ucA AUG GAC GGG GUA uuA GUA GAD
A P CCA GCC
ADD ACu AOC AAA GCU ADD GGA CAD 10 T
CGG Acu uAu GAG uAD UCG GAC UDA AAC AGU UCC GAD GAA CAG ucA COC Acu wDw GAC AGO UAU ACG UGG aOG K P D D P E L G 0 S R L L E V D N R V V V P A K CCA GAA GAU GAD CCA GAA WDG GGU CAA ucA CGU UA UuA GAA GUD GAC MAu AGA GUG GUD GUA CCA GCC AAA
12
LI
UwA
H
L
R
N
I
A
V
P
G
R
13
V
T
P
S
N
L
wUa
15200
14
A
D
V
T
S
I
P
I
P
H
S
S
V
0
V
V
E
S
S
G
V
Y
Y
G
0 CAG
K
D
Y
A
K
C
C
S
D
N
Acu CAu CuA CGU AUG ADD GUA Aca CCC GCU GAU GUA CCU CAu AGU UGG GCU GUA CCU UCC ucA GGU GOUC AAA UGU Dcc cuu UwA D
GAD K
GCU GUA CCU GGU CGO I
C
G
T
N
ucA AAD CU ACC UCC
wDa H
i
A
F
T
UCG GUA Caa
K R V G CGA GAa GGA GOD A
V
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L
uAc UAD GGU
UGC AGU 250
GAG ADD UGU GGA AcD AAU CAu GCC UD ACG CCU AuG GUG GOA GAa GCA GOG Acu UUG Aaa GAu uA GCG GAU OGG
ADOG
V
S
N
0
L
I
L
0
T
N
TER
UCC AAM CA UDA AUG CUc Caa ACC AAC UMA ACCGGGGAAGCOGAAGCGGAADGCMUCUGGGGu FIG. 1. RNA editing sites in cox2 mRNA. The unedited RNA sequence for cox2 is shown on the upper line (7). The edited RNA sequence was determined by direct RNA sequence analysis of polysomal RNA, and edited nucleotides and amino acid changes are shown beneath the unedited sequences. The RNA editing sites and the corresponding codons are identified numerically and with a box. Codon numbers are indicated above codons on the right. GUA
*
Corresponding author. 4278
VOL. 11, 1991
NOTES
EcoRI
cation (15) of unspliced exon 1 or 2 with oligonucleotides 6
BamHl -
~~~~~-0
Ofigo 6 Oligo 11
and 11 or 7 and 1, respectively (Fig. 2). PCR products were cloned into the Bluescript vector. Sequence analysis of 10 cDNA clones for each unspliced exon indicated that a spectrum of completely unedited to completely edited cDNA clones was present (Fig. 3). The genomic DNA sequence, a completely edited cDNA, and two incompletely edited cDNAs are shown in Fig. 4. The cDNA clone in Fig. 4C is processed at edit sites 10 and 11 but not at site 12; the clone in Fig. 4D is edited at site 10 and at only one of two nucleotides in site 11. The presence of incompletely edited mRNAs in the transient population of unspliced transcripts suggests that these transcripts represent intermediates in the RNA editing process (Fig. 3). Incompletely edited transcripts have been previously observed in Oenothera mitochondria, although it was uncertain whether the incompletely edited transcripts represented intermediates or an inefficiency in RNA editing (17). The cDNA clones for unspliced cox2 transcripts represent transcripts in different stages of editing (Fig. 3). The modified sites in transcripts with very few edited sites (clones 2 to 5; clones 12 to 14) suggest that the first RNA editing events may occur at any of several editing sites. The modifications in transcripts with an intermediate number of edited sites (clones 5 to 7 and clones 14 to 16) indicate that editing tended to occur in clusters that might reflect RNA editing domains. No strong directional bias is indicated by these RNA editing intermediates from maize mitochondria, in contrast to the 3'-to-5' polarity of the editing process in kinetoplast transcripts from parasitic protozoa (2, 18). The relative abundance of incompletely edited RNAs in the nascent population of unspliced cox2 transcripts suggests that RNA editing is a posttranscriptional process. The data in Fig. 3 indicate that only 61% (55 of 90) and 73% (66 of 90) of the edit sites were modified in the 10 unspliced cDNA clones for exons 1 and 2, respectively. In contrast, 99% (267 of 270) of the edit sites were modified in the 15 spliced cDNA clones of cox2 (see below). Thus, a temporal correlation exists: the nascent population of transcripts is incompletely processed with respect to both splicing and editing, but the mature population of transcripts is both spliced and virtually homogeneously edited. In addition, more than half of the cDNA clones for exon 1 were derived from incompletely edited transcripts (Fig. 3), although the cDNA synthesis was primed in the 3' untranslated region (Fig. 2). Thus, transcription of these unedited mRNAs had proceeded more than
-----MI 3 Terminus
OigO 1
Ofigo 7
FIG. 2. Cloning of spliced and unspliced exons of cox2 transcripts. The cox2 gene has two exons and a single intron. The 5' terminus of the transcript extends upstream of the EcoRI site, and the 3' terminus of the transcript is shown by the vertical arrow. Unspliced cDNAs were amplified by PCR with oligonucleotides 7 and 1 or oligonucleotides 11 and 6. Spliced cDNAs were amplified by PCR with oligonucleotides 11 and 1. Oligonucleotides represent the following nucleotide sequences (nucleotide numbers of reference 7): 1, 1641 to 1660 (specific for 3' untranslated flank); 6, 796 to 817 (intron specific); 7, 620 to 639 (intron specific); and 11, -68 to -45 (specific for 5' untranslated flank).
etiolated maize seedlings (B37N) as previously described (12) and modified with the addition of 0.2% Triton X-100, 100 ,ug of chloramphenicol per ml, 0.5 mg of heparin per ml, and 0.4 M KCI to the homogenization medium. RNA sequence analysis was performed by dideoxy-chain termination of a cDNA synthesis reaction with 32P-labeled synthetic oligonucleotides and avian myeloblastosis virus reverse transcriptase (8). Eighteen nucleotides in the coding sequence and one RNA edit site in the untranslated 5'-flanking region differ from the genomic DNA sequence (7); all sequence changes reflect a C-to-U transition. The 18 edited nucleotides in the coding sequence result in 17 changes in the amino acid sequence; edit site 11 has two edited nucleotides. Three additional edit sites in maize cox2 transcripts were previously reported as partially edited sites; none of the partially edited sites modify the amino acid sequence (4). Direct RNA sequence analysis did not indicate partial editing at these sites; however, 1 of 15 spliced cDNA clones was edited at one of these sites (codon 10, nucleotide 30). Several additional cases of editing were infrequently observed by sequence analysis of cDNA clones and were not detected by direct RNA sequence analysis (data not shown). Primary transcripts for cox2 are processed to the mature form by splicing of a single intron (7) (Fig. 2). The temporal relationship between RNA editing and intron splicing was examined by sequence analysis of cDNA clones of unspliced cox2 transcripts. Unspliced cDNAs were obtained by reverse transcription of total mitochondrial RNA with oligonucleotide 1 and polymerase chain reaction (PCR) amplifi-
1 Site#1 1
-
2 3 4 5 6 7 8 9
+ + +
10
+
+ +
--I e II 1I I I II 2 3
4
-_
_
5 6
I7 8 7
8
++ +'
+
+
++
-
++
+
++
++ ++
+
+
+
+
+ +
10
-
11
-
12 13
_ _
+ _
+
+
I
rH 9
-
+ +
4279
+ +
14 15 16 17 18 19 20
W-Col
11 11 1
11
12
13 14 15
1617
++ ++ + +++ +++ +++ +++
+++ ++ ++
+4.
++
18
+ +- + +- -
+
+ + + + +
FIG. 3. Summary of RNA editing sites in exons of unspliced cox2 transcripts. cDNAs of unspliced cox2 transcripts were prepared from total mitochondrial RNA (14), amplified by PCR, and cloned into Bluescript. Dideoxy sequence analysis of cDNA clones was performed to evaluate the degree of editing. Edited or unedited nucleotides are indicated by + or -, respectively.
4280
NOTES A. Genomic DNA
_-
_ _
_A-
-,._
_
iA
C. Unspliced RNA
A
#10
C G
-__
- aa
_-
B. Spliced RNA
-I-- cc
#11
C-
_M -'--- U
U
-
--_
A
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A
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.....-
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-_
--
IT C#
_
5
_-
D. Unspliced RNA
---
s
. u
MOL. CELL. BIOL.
Afr
_
G A T C G A T C G A T C FIG. 4. Exon sequences in RNA editing intermediates cloned from unspliced cox2 transcripts. Genomic and cDNA clones of cox2 were sequenced by chain termination with Sequence (U.S. Biochemical). (A) DNA sequence from a genomic clone; (B) DNA sequence from a completely edited and spliced cDNA; (C and D) DNA sequence from two incompletely edited and unspliced cDNAs.
G A T C
1,600 nucleotides beyond edit site 2, but the RNA sequence in the 5' region had not been completely edited. These data indicate that RNA editing is not cotranscriptional (completed simultaneously with nucleotide insertion), and a given region of the transcript may remain unedited substantially after polymerization by RNA polymerase. Although it is not possible to demonstrate a precursor-product relationship among the RNA editing intermediates and mature mRNAs by these experiments, the results are consistent with a posttranscriptional mechanism of RNA editing. Completely edited, unspliced cDNA clones were represented for each exon of cox2. Thus, splicing occurs somewhat more slowly than editing, and RNA editing may normally precede splicing in the processing of cox2 transcripts. However, incompletely edited cDNAs for spliced transcripts of cox2 are present in maize mitochondria (see below), and complete editing is not a strict prerequisite for splicing. Edit site 9 is within intron binding sequence 1 of the group II intron of cox2 (13); it is not known whether editing at this site affects splicing. The extent of RNA editing in spliced cox2 transcripts from the polysomal RNA fraction was also examined. Direct RNA sequence analyses with polysomal RNA suggested that the RNA was homogeneous, with no indication of polymorphism in the population of transcripts (summarized in Fig. 1), although low-abundance forms would not be detected by this analysis. To further examine the extent of RNA editing of spliced cox2 transcripts, cDNA clones were obtained from total maize polysomal RNA (Fig. 2). DNA sequence analysis of 15 cDNA clones indicated that most of the spliced cox2 transcripts in the polysomal RNA fraction were completely edited. Twelve cDNA clones were completely edited; however, three spliced cDNA clones were unedited at a single site (edit sites 3, 12, and 17; data not shown). If the cDNA clones were derived from transcripts that were engaged in translation, and not from nonpolysomal contamination, the incompletely edited transcripts at sites 12 and 17 would code for the synthesis of polypeptides with radical amino acid substitutions (Fig. 1). The presence of incompletely edited polysomal transcripts raises the possibility that expression of cox2 may be polymorphic in maize mitochondria.
This work was supported by an Individual Research Award in Plant Biology to R.M.M. from the McKnight Foundation. REFERENCES 1. Benne, R., J. Van den Burg, J. P. Brakenhoff, P. Sloof, J. H. Van Boom, and M. C. Tromp. 1986. Major transcript of the frameshifted cox2 gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. Cell 46:819-826. 2. Blum, B., N. Bakalara, and L. Sinpson. 1990. A model for RNA editing in kinetoplastid mitochondria: "guide" RNA molecules transcribed from maxicircle DNA provide the edited information. Cell 60:189-198. 3. Coveilo, P. S., and M. W. Gray. 1989. RNA editing in plant mitochondria. Nature (London) 341:662-666. 4. Covello, P. S., and M. W. Gray. 1990. Differences in editing at homologous sites in messenger RNAs from angiosperm mitochondria. Nucleic Acids Res. 18:5189-5196. 5. Feagin, J. E., J. M. Abraham, and K. Stuart. 1989. Extensive editing of the cytochrome c oxidase III transcript in Trypanosoma brucei. Cell 53:413-422. 6. Feagin, J. E., D. P. Jasmer, and K. Stuart. 1987. Developmentally regulated addition of nucleotides within apocytochrome b transcripts in Trypanosoma brucei. Cell 49:337-345. 7. Fox, T., and C. Leaver. 1981. The Zea mays mitochondrial gene coding cytochrome c oxidase subunit II has an intervening sequence and does not contain TGA codons. Cell 26:315-323. 8. Geliebter, J. 1988. Dideoxynucleotide sequencing of RNA and uncloned cDNA. Focus 9:5-8. 9. Gualberto, J. M., L. Lamattina, G. Bonnard, J. H. Weil, and J. M. Grienenberger. 1989. RNA editing in wheat mitochondria results in the conservation of protein sequences. Nature (London) 341:660-662. 10. Gualberto, J. M., J. H. Weil, and J. M. Grienenberger. 1990. Editing of the wheat coxIII transcript: evidence for twelve C to U and one U to C conversions and for sequence similarities around editing sites. Nucleic Acids Res. 18:3771-3776. 11. Hiesel, R., B. Wissinger, W. Schuster, and A. Brennicke. 1989. RNA editing in plant mitochondria. Science 246:1632-1634. 12. Jackson, A. O., and B. A. Larkins. 1976. Influence of ionic strength, pH, and chelation of divalent metals on isolation of polyribosomes from tobacco leaves. Plant Physiol. 57:5-10. 13. Michel, F., K. Umesono, and H. Ozeki. 1989. Comparative and functional anatomy of group II catalytic introns-a review. Gene 82:5-30. 14. Mulligan, R. M., A. P. Maloney, and V. Walbot. 1988. RNA processing and multiple transcription initiation sites result in transcript size heterogeneity in maize mitochondria. Mol. Gen.
VOL. 11, 1991 Genet. 211:373-380. 15. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. 16. Schuster, W., R. Hiesel, B. Wissinger, and A. Brennicke. 1990. RNA editing in the cytochrome b locus of the higher plant Oenothera berteriana includes a U-to-C transition. Mol. Cell. Biol. 10:2428-2431.
NOTES
4281
17. Schuster, W., B. Wissinger, M. Unseld, and A. Brennicke. 1990. Transcripts of the NADH-dehydrogenase subunit 3 gene are differentially edited in Oenothera mitochondria. EMBO J. 9:263-269. 18. Sturm, N. R., and L. Simpson. 1990. Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from Leishmania tarentolae mitochondria: RNA editing intermediates. Cell 61:871-878.
Vol. 11, No. 8
RNA Editing Intermediates of cox2 Transcripts in Maize Mitochondria AUSTIN J. YANG AND R. MICHAEL MULLIGAN* Department of Developmental and Cell Biology, University of California, Irvine, California 92717 Received 26 February 1991/Accepted 1 May 1991
Eighteen cytidines are changed to uridines in the coding sequence of transcripts for cytochrome c oxidase subunit 2 (cox2) in maize mitochondria. The temporal relationship of editing and splicing was examined in cox2 transcripts by sequence analysis of spliced and unspliced cDNAs. Cloned cDNAs of unspliced cox2 transcripts ranged from clones with no edited nucleotides to completely edited forms, while spliced cDNAs were nearly completely edited. Incompletely edited transcripts in the nascent pool of unspliced transcripts represent intermediates of the editing process. These results indicate that editing proceeds without a strong directional bias and suggest that RNA editing is a posttranscriptional process.
RNA editing has been reported in the mitochondria of several organisms (1, 5, 6), including higher plant species (3, 9, 11). The nucleotide modifications observed in plant mitochondrial transcripts are usually C-to-U transitions and occasionally U-to-C changes (3, 9-11, 16). RNA editing occurs frequently in the coding sequence and usually results in a radical change of the amino acid specified by the codon
UGAADUUGGUGAGuACUAUwUCCADDCADAAAUCAUCCDU
that restores the evolutionarily conserved sequence of the polypeptide (3, 9, 11). The RNA sequences of cox2 transcripts were determined by direct RNA sequence analysis from maize polysomal RNA (Fig. 1) and is in accord with RNA sequence analysis recently reported for maize mitochondrial cox2 transcripts (4). Total polysomal RNA was obtained from 7-day-old
UCDACUCACDCUCGGDUGGUCCUACDUCUGGUGCUGCCA
2 I I3E S FILT I A L C D A A K P N Q L I L R H L C R M G AUG ADD CUD CGU UCA UUA GAA UGU CGA UOC CUC ACA AUC GCU CUU UGU GAU GCU GCG GAA CCA DOG CAA UDA GGA WA UU 4 S
0
D
A
A
T
Is I I D L D I F F F L I H H L
K
0
G
I
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N
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G
V
L
V
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Y
S
D
Y
N
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S
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0
N
A
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P
OcU CAA GAC GCA GCA ACA CCu AUG AUG CAA GGA AuC Auu GAC UuA CAu CAC GAD AUG DD uDc Uc COC ADD CDG 5 C L V K L V R A L N H F N K 0 T N P I P Q I F V S R ADD DDG GDD UUC GUA ucA CGG AUG UUG GDD CGC GCU DDA UGG CAu UGc AAC GAG CAA Acu AAu CCA AUc CCG CAA
uwL
IDAOGG
R
I
V
H
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A
W
Y
G
L
N
T
T
7
100 P
S
V
I
P
L
F
I
A
I
I
T
I
K
A
I
G
H
S
L
T
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Y
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P
AGG ADD GDD CAD GGA Acu Acu AUc GAA ADD ADu CGG ACC Auu DDw CCu AGU GUC ADD CCA UUG DUC AUD GCU AuA P
CCA
UCG DUD GCU 9
Q CAA
I
ADD T
R
L L Y CUG DDA uAc SPLICZ SITf T
UGOG UA
Y
E
ucA AUG GAC GGG GUA uuA GUA GAD
A P CCA GCC
ADD ACu AOC AAA GCU ADD GGA CAD 10 T
CGG Acu uAu GAG uAD UCG GAC UDA AAC AGU UCC GAD GAA CAG ucA COC Acu wDw GAC AGO UAU ACG UGG aOG K P D D P E L G 0 S R L L E V D N R V V V P A K CCA GAA GAU GAD CCA GAA WDG GGU CAA ucA CGU UA UuA GAA GUD GAC MAu AGA GUG GUD GUA CCA GCC AAA
12
LI
UwA
H
L
R
N
I
A
V
P
G
R
13
V
T
P
S
N
L
wUa
15200
14
A
D
V
T
S
I
P
I
P
H
S
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V
0
V
V
E
S
S
G
V
Y
Y
G
0 CAG
K
D
Y
A
K
C
C
S
D
N
Acu CAu CuA CGU AUG ADD GUA Aca CCC GCU GAU GUA CCU CAu AGU UGG GCU GUA CCU UCC ucA GGU GOUC AAA UGU Dcc cuu UwA D
GAD K
GCU GUA CCU GGU CGO I
C
G
T
N
ucA AAD CU ACC UCC
wDa H
i
A
F
T
UCG GUA Caa
K R V G CGA GAa GGA GOD A
V
T
L
uAc UAD GGU
UGC AGU 250
GAG ADD UGU GGA AcD AAU CAu GCC UD ACG CCU AuG GUG GOA GAa GCA GOG Acu UUG Aaa GAu uA GCG GAU OGG
ADOG
V
S
N
0
L
I
L
0
T
N
TER
UCC AAM CA UDA AUG CUc Caa ACC AAC UMA ACCGGGGAAGCOGAAGCGGAADGCMUCUGGGGu FIG. 1. RNA editing sites in cox2 mRNA. The unedited RNA sequence for cox2 is shown on the upper line (7). The edited RNA sequence was determined by direct RNA sequence analysis of polysomal RNA, and edited nucleotides and amino acid changes are shown beneath the unedited sequences. The RNA editing sites and the corresponding codons are identified numerically and with a box. Codon numbers are indicated above codons on the right. GUA
*
Corresponding author. 4278
VOL. 11, 1991
NOTES
EcoRI
cation (15) of unspliced exon 1 or 2 with oligonucleotides 6
BamHl -
~~~~~-0
Ofigo 6 Oligo 11
and 11 or 7 and 1, respectively (Fig. 2). PCR products were cloned into the Bluescript vector. Sequence analysis of 10 cDNA clones for each unspliced exon indicated that a spectrum of completely unedited to completely edited cDNA clones was present (Fig. 3). The genomic DNA sequence, a completely edited cDNA, and two incompletely edited cDNAs are shown in Fig. 4. The cDNA clone in Fig. 4C is processed at edit sites 10 and 11 but not at site 12; the clone in Fig. 4D is edited at site 10 and at only one of two nucleotides in site 11. The presence of incompletely edited mRNAs in the transient population of unspliced transcripts suggests that these transcripts represent intermediates in the RNA editing process (Fig. 3). Incompletely edited transcripts have been previously observed in Oenothera mitochondria, although it was uncertain whether the incompletely edited transcripts represented intermediates or an inefficiency in RNA editing (17). The cDNA clones for unspliced cox2 transcripts represent transcripts in different stages of editing (Fig. 3). The modified sites in transcripts with very few edited sites (clones 2 to 5; clones 12 to 14) suggest that the first RNA editing events may occur at any of several editing sites. The modifications in transcripts with an intermediate number of edited sites (clones 5 to 7 and clones 14 to 16) indicate that editing tended to occur in clusters that might reflect RNA editing domains. No strong directional bias is indicated by these RNA editing intermediates from maize mitochondria, in contrast to the 3'-to-5' polarity of the editing process in kinetoplast transcripts from parasitic protozoa (2, 18). The relative abundance of incompletely edited RNAs in the nascent population of unspliced cox2 transcripts suggests that RNA editing is a posttranscriptional process. The data in Fig. 3 indicate that only 61% (55 of 90) and 73% (66 of 90) of the edit sites were modified in the 10 unspliced cDNA clones for exons 1 and 2, respectively. In contrast, 99% (267 of 270) of the edit sites were modified in the 15 spliced cDNA clones of cox2 (see below). Thus, a temporal correlation exists: the nascent population of transcripts is incompletely processed with respect to both splicing and editing, but the mature population of transcripts is both spliced and virtually homogeneously edited. In addition, more than half of the cDNA clones for exon 1 were derived from incompletely edited transcripts (Fig. 3), although the cDNA synthesis was primed in the 3' untranslated region (Fig. 2). Thus, transcription of these unedited mRNAs had proceeded more than
-----MI 3 Terminus
OigO 1
Ofigo 7
FIG. 2. Cloning of spliced and unspliced exons of cox2 transcripts. The cox2 gene has two exons and a single intron. The 5' terminus of the transcript extends upstream of the EcoRI site, and the 3' terminus of the transcript is shown by the vertical arrow. Unspliced cDNAs were amplified by PCR with oligonucleotides 7 and 1 or oligonucleotides 11 and 6. Spliced cDNAs were amplified by PCR with oligonucleotides 11 and 1. Oligonucleotides represent the following nucleotide sequences (nucleotide numbers of reference 7): 1, 1641 to 1660 (specific for 3' untranslated flank); 6, 796 to 817 (intron specific); 7, 620 to 639 (intron specific); and 11, -68 to -45 (specific for 5' untranslated flank).
etiolated maize seedlings (B37N) as previously described (12) and modified with the addition of 0.2% Triton X-100, 100 ,ug of chloramphenicol per ml, 0.5 mg of heparin per ml, and 0.4 M KCI to the homogenization medium. RNA sequence analysis was performed by dideoxy-chain termination of a cDNA synthesis reaction with 32P-labeled synthetic oligonucleotides and avian myeloblastosis virus reverse transcriptase (8). Eighteen nucleotides in the coding sequence and one RNA edit site in the untranslated 5'-flanking region differ from the genomic DNA sequence (7); all sequence changes reflect a C-to-U transition. The 18 edited nucleotides in the coding sequence result in 17 changes in the amino acid sequence; edit site 11 has two edited nucleotides. Three additional edit sites in maize cox2 transcripts were previously reported as partially edited sites; none of the partially edited sites modify the amino acid sequence (4). Direct RNA sequence analysis did not indicate partial editing at these sites; however, 1 of 15 spliced cDNA clones was edited at one of these sites (codon 10, nucleotide 30). Several additional cases of editing were infrequently observed by sequence analysis of cDNA clones and were not detected by direct RNA sequence analysis (data not shown). Primary transcripts for cox2 are processed to the mature form by splicing of a single intron (7) (Fig. 2). The temporal relationship between RNA editing and intron splicing was examined by sequence analysis of cDNA clones of unspliced cox2 transcripts. Unspliced cDNAs were obtained by reverse transcription of total mitochondrial RNA with oligonucleotide 1 and polymerase chain reaction (PCR) amplifi-
1 Site#1 1
-
2 3 4 5 6 7 8 9
+ + +
10
+
+ +
--I e II 1I I I II 2 3
4
-_
_
5 6
I7 8 7
8
++ +'
+
+
++
-
++
+
++
++ ++
+
+
+
+
+ +
10
-
11
-
12 13
_ _
+ _
+
+
I
rH 9
-
+ +
4279
+ +
14 15 16 17 18 19 20
W-Col
11 11 1
11
12
13 14 15
1617
++ ++ + +++ +++ +++ +++
+++ ++ ++
+4.
++
18
+ +- + +- -
+
+ + + + +
FIG. 3. Summary of RNA editing sites in exons of unspliced cox2 transcripts. cDNAs of unspliced cox2 transcripts were prepared from total mitochondrial RNA (14), amplified by PCR, and cloned into Bluescript. Dideoxy sequence analysis of cDNA clones was performed to evaluate the degree of editing. Edited or unedited nucleotides are indicated by + or -, respectively.
4280
NOTES A. Genomic DNA
_-
_ _
_A-
-,._
_
iA
C. Unspliced RNA
A
#10
C G
-__
- aa
_-
B. Spliced RNA
-I-- cc
#11
C-
_M -'--- U
U
-
--_
A
C #12 U 3t
A
F
-
.....-
J-~A
AT-=
u #10 U A
A/U-
5U#1C_
-_
--
IT C#
_
5
_-
D. Unspliced RNA
---
s
. u
MOL. CELL. BIOL.
Afr
_
G A T C G A T C G A T C FIG. 4. Exon sequences in RNA editing intermediates cloned from unspliced cox2 transcripts. Genomic and cDNA clones of cox2 were sequenced by chain termination with Sequence (U.S. Biochemical). (A) DNA sequence from a genomic clone; (B) DNA sequence from a completely edited and spliced cDNA; (C and D) DNA sequence from two incompletely edited and unspliced cDNAs.
G A T C
1,600 nucleotides beyond edit site 2, but the RNA sequence in the 5' region had not been completely edited. These data indicate that RNA editing is not cotranscriptional (completed simultaneously with nucleotide insertion), and a given region of the transcript may remain unedited substantially after polymerization by RNA polymerase. Although it is not possible to demonstrate a precursor-product relationship among the RNA editing intermediates and mature mRNAs by these experiments, the results are consistent with a posttranscriptional mechanism of RNA editing. Completely edited, unspliced cDNA clones were represented for each exon of cox2. Thus, splicing occurs somewhat more slowly than editing, and RNA editing may normally precede splicing in the processing of cox2 transcripts. However, incompletely edited cDNAs for spliced transcripts of cox2 are present in maize mitochondria (see below), and complete editing is not a strict prerequisite for splicing. Edit site 9 is within intron binding sequence 1 of the group II intron of cox2 (13); it is not known whether editing at this site affects splicing. The extent of RNA editing in spliced cox2 transcripts from the polysomal RNA fraction was also examined. Direct RNA sequence analyses with polysomal RNA suggested that the RNA was homogeneous, with no indication of polymorphism in the population of transcripts (summarized in Fig. 1), although low-abundance forms would not be detected by this analysis. To further examine the extent of RNA editing of spliced cox2 transcripts, cDNA clones were obtained from total maize polysomal RNA (Fig. 2). DNA sequence analysis of 15 cDNA clones indicated that most of the spliced cox2 transcripts in the polysomal RNA fraction were completely edited. Twelve cDNA clones were completely edited; however, three spliced cDNA clones were unedited at a single site (edit sites 3, 12, and 17; data not shown). If the cDNA clones were derived from transcripts that were engaged in translation, and not from nonpolysomal contamination, the incompletely edited transcripts at sites 12 and 17 would code for the synthesis of polypeptides with radical amino acid substitutions (Fig. 1). The presence of incompletely edited polysomal transcripts raises the possibility that expression of cox2 may be polymorphic in maize mitochondria.
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17. Schuster, W., B. Wissinger, M. Unseld, and A. Brennicke. 1990. Transcripts of the NADH-dehydrogenase subunit 3 gene are differentially edited in Oenothera mitochondria. EMBO J. 9:263-269. 18. Sturm, N. R., and L. Simpson. 1990. Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from Leishmania tarentolae mitochondria: RNA editing intermediates. Cell 61:871-878.
