From the
In neurons of the mammalian brain primary transcripts of genes
encoding subunits of glutamate receptor channels can undergo RNA
editing, leading to altered properties of the transmitter-activated
channel. Editing of these transcripts is a nuclear process that targets
specific adenosines and requires a double-stranded RNA structure
configured from complementary exonic and intronic sequences. We show
here that the two independent editing sites in
RNA editing is a posttranscriptional process that changes the
sequence of gene transcripts
(1, 2) . This process is
common in mitochondria of plants and protozoa where, as a rule,
transcripts undergo multiple editing events to acquire translatable
open reading frames. By contrast, RNA editing has been found in only a
few mammalian nuclear transcripts where it generates single codon
substitutions and, hence, changes in the cognate proteins.
Apolipoprotein B (apoB) RNA of the small intestine provided the first
example of editing in a nuclear transcript
(3, 4) . In
this RNA a premature stop codon is introduced by cytidine deamination
(CAA to UAA), leading to a truncated protein with altered function. A
uridine to cytidine substitution changing a leucine to a proline codon
occurs in transcripts of the Wilm's tumor susceptibility gene in
kidney and testis
(5) .
Adenosine-specific RNA editing was
found in mammalian transcripts of neural origin
(6, 7, 8) . In the central nervous system the
principal excitatory neurotransmitter
L-glutamate activates
postsynaptically located receptor channels (GluRs),
Our present understanding concerning mechanistic
aspects of this enigmatic control over functional channel determinants
in central neurons is rudimentary. One recent advance has been the
identification of intronic editing site complementary sequences (ECS)
that form a short intramolecular dsRNA structure with the to-be-edited
exonic sequence
(10) . As documented for both the Q/R and R/G
sites in GluR-B pre-mRNA these dsRNA structures serve as substrates for
a positionally precise RNA editing
(8, 10) . It has been
proposed that the edited adenosines might constitute inosines generated
by adenosine deamination in RNA
(2, 6) . Indeed, the
preferred base pairing properties of inosine with cytidine would lead
to the incorporation of guanosine in a cDNA position occupied by
inosine in the RNA template. The requirement for dsRNA and the fact
that only adenosines are targeted in GluR pre-mRNAs have given rise to
the hypothesis that dsRNA adenosine deaminase might operate the
nucleotide change by hydrolytic deamination of adenosine to inosine
(2, 10, 11) .
We have now employed an in
vitro system to determine the nature of the edited adenosine in
GluR-B pre-mRNA. As shown in this study, editing by nuclear extract
displays the positional accuracy and all sequence requirements
previously established in cellular assays
(8, 10) .
Importantly, nuclear extract from HeLa cells mediates the
site-selective conversion of adenosine to inosine in both the Q/R site
and the R/G site.
Nuclear extract prepared from HeLa cells
(12) was
analyzed for RNA editing activity. RNAs transcribed in vitro from editing competent as well as incompetent GluR-B minigenes for
both the Q/R
(10) and the R/G
(8) site were incubated
with nuclear extract. Site-selective editing was determined by primer
extension (Fig. 1) performed on the recovered RNAs (Q/R site) or on
their RT-PCR products (R/G site). RNAs from minigenes (B13, Q/R site;
E2, R/G site) edited in transfected cells
(8, 10) were
also edited by the nuclear extract, whereas RNAs from minigenes
rendered editing incompetent by nucleotide deletions (B13
To
determine the chemical nature of the edited adenosine, 3`
Thus, nuclear extracts can be employed to characterize the
biochemical machinery responsible for specific adenosine editing. As a
first milestone, this in vitro system permitted the
identification of edited adenosines in GluR-B pre-mRNAs as inosine
residues. Formation of inosine is most likely due to enzymatic
adenosine deamination since upon dialysis of nuclear extract editing
efficiencies remained unchanged, indicating that nucleotides required
for transglycosylation or nucleotide replacement are not necessary for
GluR editing. Our results are compatible with the notion
(2, 10, 21) that dsRNA adenosine deaminase is a
candidate enzyme for the selective adenosine editing in GluR
transcripts of central neurons. In view of the ubiquitous expression of
this recently cloned
(21, 22) enzyme, adenosine
deamination should be more widespread in nuclear pre-mRNAs than
anticipated on the basis of GluR transcript editing. This conclusion is
further strengthened by the observation that many cell lines, including
HeLa cells, contain the machinery for the correct editing of GluR-B
pre-mRNA even in the absence of endogenous GluR gene expression
(10) . We now anticipate the unprecedented occurrence of inosine
in select codons of other mammalian mRNAs. As established for GluRs,
these codons
(23) are likely to specify functionally critical
residues in the cognate protein products. Hence, we propose that
site-selective adenosine deamination may be a general mechanism for
increasing the functional diversity of mammalian gene products.
All reactions (25 µl) contained 12.5 µl of
nuclear extract and were incubated for 3 h at 30 °C. Numbers in
parentheses indicate independent determinations; standard deviations of
values listed were
We thank Annette Herold for DNA sequencing, Andrea
Bürer (Basel) for cell culture and preparation of nuclear
extracts, and Jutta Rami for assistance with the manuscript. The help
of Drs. Mario Mörl and Rainer Frank and the critical input by our
colleagues Drs. Rolf Sprengel, Mary O'Connell, Hartmut
Lüddens, and Thomas Kuner are gratefully acknowledged.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor
GluR-B pre-mRNA are edited with positional accuracy by nuclear extract
from HeLa cells. Nucleotide analysis by thin layer chromatography of
the edited RNA sequences revealed selective adenosine to inosine
conversion, most likely reflecting the participation of double-stranded
RNA adenosine deaminase. Our results predict the presence of
inosine-containing codons in other mammalian mRNAs.
(
)
the subunits of which form an extended gene family,
comprising 16 members to date
(9) . In this family, the
transcripts of five genes are known to undergo RNA editing in a total
of eight positions. In each instance, the transmitter-activated channel
acquires altered ion permeability or kinetic properties
(8, 9) . GluR transcript editing exclusively targets
gene-specified adenosines that appear as guanosines in cloned cDNA. For
example, the
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
receptor GluR-B pre-mRNA is edited in two independent positions, from a
CAG to a CGG codon (Q/R site; Ref. 6) and from an AGA to a GGA codon
(R/G site; Ref. 8).
DNA Constructs and RNA Synthesis
RNAs were
transcribed from murine GluR-B minigene constructs. For Q/R site
editing DNA constructs were B13 and B136, which carries a deletion
in its ECS sequence and hence is not edited in transfected cells
(10) . DNAs were linearized with XbaI and upon
transcription in vitro generated RNAs of an approximate length
of 720 nucleotides, including all of exon 11 and 530 nucleotides of
intron 11. For R/G site editing, DNA constructs were
BglII- BglII (exon 13 into intron 15) carrying the
intronic E2 mutation (increased editing relative to wild type sequence)
and BglII- BglII carrying the intronic S1 mutation
(editing incompetent)
(8) . These constructs were linearized
with StuI (unique site in exon 14) and yielded RNAs of
approximately 1,100 nucleotides containing exon 13, intron 13, and part
of exon 14. In vitro transcription was performed for 60 min at
37 °C in 25 µl containing 80 m
M Hepes-KOH, pH 7.5, 32
m
M MgCl
, 2 m
M spermine, 40 m
M DTT, 5 m
M each of ATP, GTP, CTP, and UTP, 10 µCi of
-[
P]GTP (3,000 Ci/mmol), 750 units of SP6
RNA polymerase (Promega), 37.5 units of RNasin (Promega), 475 units of
inorganic pyrophosphatase from yeast (Promega), and 1 µg of
linearized recombinant vector DNA. Radiolabeled, high specific activity
RNAs were synthesized in the presence of 10 µ
M GTP and 120
µCi of
-[
P]GTP (Amersham Corp., 3,000
Ci/mmol). Unincorporated nucleotides were removed by EtOH precipitation
of the phenol/chloroform-extracted reactions. RNAs used for reverse
transcription (RT) followed by PCR
(12) were treated three
times for 45 min at 37 °C with 10 units of RNase-free DNase
(Boehringer Mannheim) in the presence of 20 units of RNasin
(Fermentas). Removal of DNA from RNA was demonstrated by PCR
amplification (see below).
RNA Editing in Nuclear Extract
Nuclear extracts
were prepared from HeLa cells according to Ref. 13 except that high
salt buffer C contained 1.2
M KCl instead of NaCl. Extracts
were stored in aliquots at -80 °C and had a protein
concentration of about 10 µg/µl in 10 m
M Tris-HCl, pH
7.9, 12.5% glycerol, 0.75 m
M MgCl, 0.1 m
M EDTA, and approximately 270 m
M KCl, as measured by
conductivity. For standard assays, RNAs (between 10 and 100 fmol) were
incubated at 30 °C for 3 h in a 25-µl reaction mixture
containing 12.5 µl of nuclear extract and having a final buffer
composition (``Q buffer'') of 25 m
M Tris-HCl, pH
7.9, 135 m
M KCl, 2.5 m
M EDTA, 6.25% glycerol, 1
m
M DTT, 50 µg/ml poly(A). Reactions (25 µl) having 2
µl of nuclear extract yielded in B13 and E2 RNAs approximately half
of the editing levels achieved with 12.5 µl of extract. After
incubation, reaction mixtures were deproteinized by adding equal
volumes of 200 m
M Tris-HCl, pH 7.9, 300 m
M NaCl, 25
m
M EDTA, 2% SDS, 0.8 mg/ml proteinase K for 30 min at 37
°C, and RNA was recovered by phenol/chloroform extraction and
ethanol precipitation. Preincubation of extract with 1,000 units/ml
micrococcal nuclease (Pharmacia Biotech Inc.) for 30 min at 30 °C
did not reduce Q/R site editing; incubation was in the presence of 1
m
M CaCl
, which was chelated by 2 m
M EGTA
before adding B13 RNA. Preincubation (30 min, 30 °C) with
proteinase K (0.8 mg/ml) abolished Q/R site editing.
Analysis by Primer Extension
For Q/R site editing,
10 fmol of 5` P-labeled oligonucleotide primer B-RT (3
10
dpm/pmol) were hybridized to extract-incubated
and purified RNA (approximately 3-5 fmol) in 50 m
M Tris-HCl, pH 8.3, 75 m
M KCl, 3 m
M MgCl
, 10 µ
M each of dATP, dGTP, and dCTP
and 250 µ
M ddTTP (Pharmacia) by heating for 5 min to 80
°C and annealing at 55 °C for 4 h in a final volume of 9
µl. After addition of 0.8 units of avian myeloblastosis virus
reverse transcriptase (Life Technologies, Inc.) and 1 µl of 0.1
m
M DTT the reactions (10.5 µl final volume) were incubated
for 1 h at 42 °C. For the analysis of RT-PCR products generated
from extract-incubated R/G-edited RNAs, approximately 20 ng of agarose
gel-purified PCR product was denatured in alkaline,
ethanol-precipitated, and resuspended in 50 m
M Tris-HCl, pH
8.3, 75 m
M KCl, 3 m
M MgCl
, 10 µ
M each of dATP, dGTP, dCTP, 250 µ
M ddTTP, and 10 fmol
of the
P-labeled oligonucleotide B-RTFF45. After 5 min at
55 °C, 0.8 unit of avian myeloblastosis virus reverse transcriptase
plus 1 µl of 0.1
M DTT were added, and the reactions (10.5
µl final) were incubated for 45 min at 42 °C. Nucleic acid was
fractionated on a 15% polyacrylamide, 7
M urea gel, and dried
gels were exposed to x-ray film with a DuPont Cronex Lightning
intensifying screen. Quantitative analysis was performed on a
PhosphorImager (Fuji, BAS1000).
Analysis by Cloning
Extract-incubated and purified
RNAs (3-5 fmol), resuspended in 20 µl of HO
containing 1 µ
M primer for reverse transcription (KMH3 for
Q/R site editing, BFFK3 for R/G editing), were denatured (70 °C, 10
min) and added to a 10-µl RT mix (50 m
M Tris-HCl, pH 8.5,
75 m
M KCl, 3 m
M MgCl
, 10 m
M DTT,
20 units of RNasin, and 500 µ
M of each dNTP). A 10-µl
aliquot of each reaction was removed for control purposes (``mock
RT''). The remainder was incubated with 200 units of reverse
transcriptase (Moloney murine leukemia virus, Life Technologies, Inc.)
for 1 h at 37 °C and 5 min at 95 °C. A first PCR amplification
using the primers lacZ1/PCRK3 (Q/R site editing) or cis55/PCRK3 (R/G
site editing) was in 20 µl of 20 m
M Tris-HCl, pH 8.4, 50
m
M KCl, 1.5 m
M MgCl
, 200 µ
M dNTPs, 400 n
M primers, and 0.5 unit of Taq polymerase (Life Technologies, Inc.). Cycle conditions were: 94
°C, 3 min; 20 step cycles (94 °C, 20 s; 55 °C, 30 s; 72
°C, 40 s); and 72 °C, 10 min. A second PCR amplification with
the nested primer sets MH36/lacZ1 (Q/R site editing) or cis55/intB1
(R/G site editing) was in 50 µl for 35 step cycles. The
PCR-generated DNA fragments served as templates for primer extension at
the R/G site or were directionally cloned in doubly digested phage M13
RF-DNA (M13 mp19, cleaved with PstI and EcoRI for Q/R
site fragments; M13 mp18, cleaved with KpnI and EcoRI
for R/G site fragments)
(14) . Filter lifts of recombinant phage
plaques were hybridized overnight at 22 °C in 5
SSC (1
SSC: 0.15
M NaCl, 15 m
M sodium citrate) with
P-labeled oligonucleotides B-R (Q/R site) or EDFF-3 (R/G
site). Filters were washed in 1
SSC at 56 °C (Q/R site) or
50 °C (R/G site) to distinguish edited from unedited sequences. At
least 12 recombinant phage DNAs carrying edited and unedited Q/R and
R/G forms were sequenced (ABI sequencer 373A).
Base Modification
RNAs (150 fmol, 10
dpm/µg) incubated in nuclear extract were purified (see
above) and resuspended in 20 µl of 10 m
M PIPES, pH 6.4,
100 m
M NaCl, 0.25 m
M EDTA containing 50 pmol of the
oligonucleotides Bprotect (Q/R site) or FF-Bprotect (R/G site).
Oligonucleotide was annealed to RNA (3 min, 90 °C; 30 min, 55
°C), and 170 µl of 10 m
M Tris-HCl, pH 7.5, 300 m
M NaCl, 5 m
M EDTA containing 0.2 unit of RNase A and 70
units of RNase T1 (Boehringer Mannheim) were added. RNase digestion was
for 30 min at 37 °C (Bprotect) or 32 °C (FF-Bprotect), followed
by phenol/chloroform extraction and ethanol precipitation with 10
µg of glycogen as carrier. Precipitated nucleic acid was
resuspended in 10 µl of 0.1% bromphenol blue, 0.1% xylene cyanol
and electrophoresed on a native 15% polyacrylamide gel in 1
TBE
(90 m
M Tris borate, 2 m
M EDTA, pH 8.0) at 200 V for
8-9 h. Protected bands were gel-eluted overnight at 22 °C in
0.5
M NH
Ac, 1 m
M MgCl
, 0.1
m
M EDTA, 0.1% SDS. Nucleic acid was collected by ethanol
precipitation after sterile filtration (Millipore, 0.22
µ
M) of the eluate, denatured (5 min, 70 °C), and
digested to 3`-nucleoside monophosphates in 20 µl of 10 m
M NH
Ac, pH 4.5, containing 0.5 unit of ribonuclease T2
(Life Technologies, Inc.) for 4 h at 37 °C. The digest was dried in
a SpeedVac concentrator, resuspended in 3 µl of water containing
3`-XMPs (where X is A, I, G, C, or U; 1 µg/µl each; Sigma),
spotted on a cellulose plate (Sigma), and chromatographed in saturated
(NH
)
SO
, 0.1
M NaAc, pH 6,
2-propyl alcohol (79:19:2, by volume).
Quantification of 3`-XMPs
Internal labeling of RNA
by -[
P]GMP combined with ribonuclease
T2-mediated label transfer permitted an evaluation of label
distribution in 3`-XMPs derived from the oligonucleotide-protected RNA
moieties. In the protected B13 RNA moiety (Fig. 2 a), the
5`-
P groups of nine GMPs are transferred by T2 to three
3`-GMPs, three 3`-UMPs, two 3`-AMPs (unedited), or one 3`-IMP (fully
edited). Within the E2 RNA moiety (Fig. 2 b) these
numbers are five 3`-GMPs, two 3`-UMPs, one 3`-AMP (unedited), or one
3`-IMP (edited). Radioactive spots on one-dimensional TLCs (3`-UMP and
3`-CMP co-migrate) were quantified with a PhosphorImager (Fuji, BAS
1000), and the relative abundance of each 3`-XMP was determined. In
four experiments the averaged ratios ± S.D. for the labeled
3`-XMPs from protected B13 RNA were G/C + U/A/I, 2.6 ±
0.1/3.2 ± 0.1/2.1 ± 0.1/0.11 ± 0.06 (theoretical
values: 3/3/1.7/0.3, assuming 30% Q/R site editing) and for E2, 4.1
± 0.3/2.1 ± 0.2/1.6 ± 0.2/0.2 ± 0.1
(theoretical: 5/2/0.9/0.1, assuming 10% R/G site editing). Deviations
from predicted values result from contamination by heterodisperse RNA
fragments of the gel-isolated heteroduplexes, as evidenced by
radioactive material in gel lanes resolving the unprotected
ribonuclease A and T1-treated RNAs (Fig. 2) and the
3`-[
P]CMP spot on two-dimensional TLC
(Fig. 3).
Figure 2:
In vitro editing of GluR-B pre-mRNA
generates inosine from adenosine. a, B13 RNA spans GluR-B exon
11 and the proximal part of intron 11 (Ref. 10). b, E2 RNA
contains GluR-B exon 13, intron 14, and part of exon 14 (Ref. 8). The
Q/R and R/G editing sites and the intronic ECS sites are indicated by
open arrowheads. After incubation in nuclear extract
the recovered RNAs were hybridized to the oligonucleotide Bprotect
( a) or FF-Bprotect ( b). Excess RNA was removed by
ribonucleases A and T1 to generate the heteroduplexes shown. The
asterisks indicate the 5`-P group carried by GMP
residues in the RNA moiety of each heteroduplex. The
oligodeoxynucleotide-protected RNAs were isolated by gel
electrophoresis. Lanes 1 in a,
P-end labeled Bprotect hybridized to excess synthetic RNA
oligomer having the unedited GluR-B Q/R site sequence. All other
lanes for both a and b show material
digested by ribonuclease A and T1. lane 2, digested material
of lane 1; lane 3, B13 RNA protected by
oligonucleotide; lane 4, unprotected B13 RNA; lane 5,
protected B13
6 RNA; lane 6, unprotected B13
6 RNA;
(b) 1, protected E2 RNA; 2, unprotected E2 RNA; 3, protected S1 RNA; 4,
unprotected S1 RNA. Gel-isolated heteroduplexes (for gel positions see
brackets) were heat-denatured, subjected to ribonuclease T2 digestion,
and 3`XMPs were resolved by one-dimensional TLC. Lanes (a) 1,
extract-incubated, oligodeoxynucleotide-protected B13 RNA; 2,
extract-incubated, unprotected B13 RNA; 3, Q-buffer-incubated,
protected B13 RNA; 4, Q-buffer-incubated, unprotected B13 RNA; 5,
extract-incubated, protected B13
6 RNA; 6, extract-incubated,
unprotected B13
6 RNA; (b) 1, extract-incubated,
oligodeoxynucleotide-protected E2 RNA; 2, extract-incubated, protected
S1 RNA; 3, Q-buffer-incubated, protected E2 RNA. Migration standards
(3`XMPs: Cp+Up, Ip, Gp, Ap) and origin are
indicated.
Figure 3:
Two-dimensional thin-layer chromatography
of ribonuclease T2 digestion products of the
oligodeoxynucleotide-protected in vitro edited B13
( a) and E2 ( b) RNAs. Isobutyric
acid/NHOH/water (66:1:33, by volume) was used as solvent
for the first dimension (migration, bottom to top)
and saturated (NH
)
SO
, 0.1
M NaAc pH 6, 2-propanol (79:19:2, by volume) for the second
dimension (migration, left to right). The individual
3`-XMPs (Gp, Ap, Ip, Up, Cp) are indicated on the chromatograms. The
presence of 3`-[
P]CMP is likely to reflect a
contamination by heterodisperse RNA fragments of the protected,
gel-isolated RNAs (Fig. 2).
Oligonucleotides Used in This Study
B-RT,
5`-GGCGAAATATCGCATCCTTG-3`, antisense, GluR-B, exon 11; B-RTFF45,
5`-ATTGTTATACTATTCCACCC-3`, antisense, GluR-B, intron 13; Bprotect,
5`-CATCCTTGCCGCATAAAGGCACCC-3`, antisense, GluR-B, exon 11,
complementary to edited Q/R site; FF-Bprotect,
5`-CCACCCACCCTAATGAGGATCC-3`, antisense, GluR-B, intron 13/exon 13
border, complementary to edited R/G site; KMH3,
5`-GACACGGTACCACACAACGGCATTTCCATGAATTGATGTTAGAG-3`, revT primer,
anti-sense, GluR-B, intron 11;
BFFK3,5`-GACACGGTACCACACAACGGATTGTGAGTTACCTCATATCCG-3`, revT primer,
antisense, GluR-B, intron 13; lacZ1,
5`-GCCTGCAGCCATGGTGAATCAACTAACGAATTTGG-3`, sense, GluR-B, exon 11;
PCRK3, 5`-GACACGGTACCACACAACGG-3`, PCR primer for cDNA primed with KMH3
or BFFK3; cis55, 5`-CTCTGCGAGCTCAGGTCCAACTGCACCTCGG-3`, vector-specific
5` primer; MH36, 5`-TCACCAGGGAAACACATGATCAAC-3`, antisense, GluR-B,
intron 11; intB1, 5`-GCGGTACCGTGAGTTACCTCATATCCGTAT-3`, antisense,
GluR-B, intron 13; B-R, 5`-GCATCCTTGCCGCATAAAGGC-3`, antisense, GluR-B,
exon 11, complementary to edited Q/R site; EDFF-3,
5`-CCACCCTAATGAGGATCCTT-3`, antisense, GluR-B, intron 13/exon 13
border, complementary to edited R/G site.
6, Q/R
site) or substitutions (S1, R/G site) in the intronic ECS site were not
(Fig. 1, ). The extent of site-specific editing was
time-dependent and, after 3 h, reached (mean ± S.D.) 32 ±
6% ( n = 10) for the Q/R site and 10 ± 2% ( n = 8) for the R/G site (Fig. 1, ). These
editing efficiencies were confirmed by an assay
(10) based on
PCR-mediated sequence amplification
(13) , directional cloning
of amplified DNAs, and probing the recombinant phage plaques with
oligonucleotides for edited and unedited sequence forms. Furthermore
and importantly, DNA sequencing of sets of recombinant phage DNAs
derived from RNAs incubated in nuclear extract (Fig. 1, legend)
demonstrated that the only edited positions concerned the adenosines of
the Q/R and R/G sites (not shown). Collectively these data indicate
that the sequence requirements for the in vitro editing of two
independent positions in GluR-B pre-mRNAs are those previously
established by cell transfection. Similarly, the positional selectivity
of editing observed for GluR-B minigenes in transfected cells is
strictly maintained by the nuclear extract. Analysis of 60 cloned DNA
sequences derived from in vitro edited B13 RNA showed no
evidence for the low level editing of additional adenosines observed
around the Q/R site in native GluR-B pre-mRNA
(10) .
Figure 1:
Primer extension assays.
a, GluR-B cDNA is shown on top with the Q/R
(nucleotide position 1820, amino acid residue 607) and R/G (nucleotide
position 2290, amino acid residue 764) sites indicated by open arrowheads. M1 to M4 ( filled boxes) indicate the four predicted transmembrane
segments; F/F ( hatched box) depicts the
alternatively spliced exon Flip or Flop (24, 25). The nucleotide
sequences of GluR-B pre-mRNA around the Q/R (exon 11) and R/G (exon 13)
sites are shown below and are contained on B13 RNA (Q/R site) and E2
RNA (R/G site). For the R/G site this sequence is in part intronic
( lowercase letters) due to close apposition of the
editing-targeted adenosine to the 5` splice site of intron 13. The
location of the P-5`-end labeled primers B-RT (Q/R site)
and B-RTFF45 (R/G site) are indicated below the partial sequences of
B13 RNA and below the RT-PCR product of E2 RNA. Primer extension in the
presence of ddTTP resulted in two extension products for each editing
site, which differed in length for the unedited and edited sequences.
rev T, reverse transcription. The products were resolved on
polyacrylamide gels ( b), and the relative signal intensities
of the DNA bands were used to evaluate the percentage of site-selective
adenosine editing. Lane 1, B13 RNA incubated in
Q-buffer for 5.5 h; lanes 2-7, B13 RNA in nuclear
extract for 0, 0.5, 1, 2, 3, and 5.5 h; lane 8, B13
6 RNA
in extract for 5.5 h; lane 9, E2 RNA in Q-buffer for 5.5 h;
lanes 10-14, E2 RNA in extract for 0, 1, 2, 3, and 5.5
h; lane 15, S1 RNA in extract, 5.5 h. c, time course
of site-selective adenosine editing in vitro for B13 and E2
RNAs and their ECS mutants B13
6 and S1.
Editing
was abolished by heat treatment (10 min, 65 °C) or proteinase K
digestion of nuclear extract but not by micrococcal nuclease. GluR-B
pre-mRNAs when incubated in the absence of extract under conditions
used to reveal RNA self-modification
(15) showed no
site-selective editing. Thus, editing appears not to be mediated by the
RNA itself nor does it require other RNAs but is mediated by protein
components provided by the nuclear extract. Indeed, the extent of
editing depended on the amount of extract (see ``Materials and
Methods''). Failure of the editing reaction to go to completion
did not appear to result from limiting amounts of editing factor(s) in
extract since editing efficiencies remained constant over a 10-fold
concentration range of RNA (). The addition of ATP to 100
µ
M did not alter the extent of editing (not shown),
indicating that editing is not dependent on ATP as an energy source.
Editing was not affected by poly(I), poly(C), and poly(dI)poly(dC)
but was efficiently inhibited by poly(I)
poly(C) (inhibitory
constant, IC
0.3 n
M), revealing the
participation of a factor with high affinity for dsRNA. This factor is
likely to be dsRNA adenosine deaminase
(2, 11) , which
converts adenosines to inosines in extended dsRNA with limited
positional specificity
(16) . dsRNA adenosine deaminase displays
such high affinity binding to dsRNA (equilibrium binding constant,
K
0.2 n
M; Ref. 17), and the
optimal KCl concentration (approximately 100 m
M, )
for in vitro editing of the Q/R site in GluR-B pre-mRNA was
that required by purified dsRNA adenosine deaminase
(18) for
optimal adenosine to inosine conversion in dsRNA. However, B13 RNA is
not edited by the purified enzyme,
(
)
suggesting
that site-selective editing may involve additional factor(s).
P-labeled mononucleosides (XMPs) generated by ribonuclease
digestion of a short sequence within internally labeled,
extract-incubated RNAs were analyzed by TLC
(19) . Different
GluR-B-specific RNAs were enzymatically synthesized in the presence of
-[
P]GTP and were incubated with nuclear
extract. The radiolabeled, edited RNAs, recovered intact as judged by
gel migration (not shown), were hybridized with a molar excess of an
oligodeoxynucleotide (24-mer) complementary to the RNA sequence around
the edited nucleotide position (Fig. 2). After digestion with the
single-stranded specific ribonucleases A and T1 the resultant RNA-DNA
heteroduplexes were resolved by native polyacrylamide gel
electrophoresis. The heteroduplex containing the protected B13 RNA
moiety appeared as three closely spaced bands of different intensities.
The same triplet of bands was generated by ribonuclease digestion of a
synthetic oligomeric RNA-DNA hybrid (Fig. 2 a) and
probably reflects the removal by ribonuclease of terminal bases from
the oligodeoxynucleotide-protected RNA. Gel-recovered heteroduplexes
were subjected to digestion by ribonuclease T2, which generates 3`-XMPs
from RNA
(20) , leading to the transfer of the 5`-
P
group in GMPs to the 3` position of the preceding nucleoside. Digested
material was resolved by one-dimensional TLC. As a result, radiolabel
co-migrating with 3`-IMP was readily detected in the appropriate in
vitro edited RNA species (Fig. 2). The nature of the inosine
in the edited B13 and E2 RNAs was further confirmed by two-dimensional
TLC (Fig. 3). In four experiments editing levels as assessed by
3`-[
P]IMP/3`-[
P]AMP
ratios on one-dimensional TLC were 11 ± 0.6% for B13 RNA and
13.2 ± 0.9% for E2 RNA. The low level editing of B13 RNA in
these experiments is apparently due to sequence contamination of the
gel-isolated B13 heteroduplex, as evidenced by the
3`-[
P]CMP spot on the two-dimensional
chromatogram (Fig. 3) (see also ``Materials and
Methods''). The sequence specificity of in vitro adenosine to inosine conversion was clearly demonstrated by the
absence of inosine in B13
6 RNA (Fig. 2 a) in a B13
RNA mutant in which a guanosine residue had been substituted for the
Q/R site adenosine (not shown) and in S1 RNA (Fig. 2 b).
Table:
RNA editing of GluR-B RNAs by nuclear extract
from HeLa cells
20%. ND, not determined.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.