(Received for publication, December 11, 1995; and in revised form, February 28, 1996)
From the
Pre-mRNAs for brain-expressed ionotropic glutamate receptor subunits undergo RNA editing by site-specific adenosine deamination, which alters codons for molecular determinants of channel function. This nuclear process requires double-stranded RNA structures formed by exonic and intronic sequences in the pre-mRNA and is likely to be catalyzed by an adenosine deaminase that recognizes these structures as a substrate. DRADA, a double-stranded RNA adenosine deaminase, is a candidate enzyme for L-glutamate-activated receptor channel (GluR) pre-mRNA editing. We show here that DRADA indeed edits GluR pre-mRNAs, but that it displays selectivity for certain editing sites. Recombinantly expressed DRADA, both in its full-length form and in an N-terminally truncated version, edited the Q/R site in GluR6 pre-mRNA and the R/G site but not the Q/R site of GluR-B pre-mRNA. This substrate selectivity correlated with the base pairing status and sequence environment of the editing-targeted adenosines. The Q/R site of GluR-B pre-mRNA was edited by an activity partially purified from HeLa cells and thus differently structured editing sites in GluR pre-mRNAs appear to be substrates for different enzymatic activities.
The alteration of codons by RNA editing, leading to changes in
protein structure and function, represents a newly recognized type of
posttranscriptional modification in mammalian nuclear transcripts and
occurs by site-specific base modification(1, 2) . In
the transcript for intestinal apolipoprotein B (apoB), ()a
translational stop codon is generated by cytidine deamination,
generating the expression of a truncated protein with altered function (1) . By contrast, specific adenosines are deaminated (2, 3) in pre-mRNAs for subunits of glutamate-gated
receptor channels (GluR) (4) . At the Q/R site (5) of
the
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)
receptor subunit GluR-B and the high-affinity kainate receptor subunits
GluR5 and GluR6, a glutamine codon CAG is converted to the arginine
codon CIG. At the R/G site of the AMPA receptor subunits GluR-B, -C,
and -D (6) an arginine codon AGA is switched to the glycine
codon IGA, and at the I/V and Y/C sites in GluR6, an ATT codon is
changed to ITT and a TAC to TIC, respectively(7) . Each of the
amino acid changes generated by RNA editing alters functional
properties of the glutamate-activated channel(3, 4) .
Different from apoB RNA editing(1) , the site-specific adenosine deamination in GluR transcripts requires a double-stranded (ds)RNA structure formed by the exonic sequence around the editing site and an intronic editing site complementary sequence (ECS)(3, 8) , predicting that this type of RNA editing is catalyzed by an adenosine deaminase that operates on dsRNA. In addition to exonic adenosines, some intronic adenosines are also converted, including hotspot1 in GluR-B intron 11 (8) and in GluR6 pre-mRNA(9) . The site-selective adenosine to inosine conversion in GluR-B pre-mRNA could be demonstrated in vitro(10, 11, 12) . dsRAD(2, 13) , also termed DRADA(14) , is a dsRNA-specific adenosine deaminase that is widely expressed, both with respect to species and tissue. This enzyme lacks site-selective activity on extended dsRNAs but displays a sequence-dependent modification of specific adenosines in short synthetic dsRNAs(15) . Although cloned human (16) and rat (17) cDNAs for DRADA have been isolated, the physiological substrates for this enzyme have yet to be identified. DRADA is currently viewed as a candidate enzyme for GluR pre-mRNA editing(2, 14) , but this view lacks experimental support.
We now demonstrate that DRADA is indeed capable of editing specific adenosines in GluR pre-mRNAs in vitro. Indicating substrate selectivity for certain editing sites, the recombinantly expressed deaminase edited in synthetic pre-mRNAs the R/G site of GluR-B and the Q/R site of GluR6, but not the Q/R site of GluR-B. This latter site appears to be the substrate for a different activity, as indicated by fractionation of nuclear extract from HeLa cells. A comparison of the dsRNA structures for the different sites suggests that the structural environment of the to-be-edited adenosine may be one determinant for the substrate selectivity by different editing activities.
Figure 1:
Recombinant DRADA edits
selectively some sites in GluR pre-mRNAs. A, schematic
representation of a GluR subunit(4) , its pre-mRNA, and the
dsRNA structure of exonic and intronic sequences (6, 8, 9) as a substrate for site-selective
adenosine deamination. A GluR subunit is depicted from N to C terminus
with the four black boxes denoting segments for membrane
insertion(4) . X/Y indicates alternative amino acid
residues, one (X) gene-encoded and the other (Y)
introduced by site-selective RNA editing. Shown below is the pre-mRNA
segment around the region containing the exonic editing site (ES) and the intronic ECS element essential for site selective
RNA editing. Exonic and intronic RNA sequences form a dsRNA structure
as schematically indicated, with the adenosine targeted for deamination
by a dsRNA-dependent adenosine deaminase shown in bold. B and C, dependence of editing at four sites in GluR pre-mRNAs on
the amount of recombinant DRADA in its full-length (wt) form (B) and an N-terminally truncated 88 kDa form (C) (N5, see Fig. 2). The amount of enzyme is
indicated in units determined by adenosine conversion on extended dsRNA
(see ``Materials and Methods''). D, Western blot
analysis with anti-FLAG antibody of purified recombinant DRADA,
full-length (wt) and N-terminally truncated (
N5). E, primer extension analysis of RT-PCR products from in
vitro edited GluR6(Q/R) pre-mRNA. The exonic sequence around the
editing site (nucleotides A/G) is shown on the left. The
correspondence to adenosines of gel bands containing primer extension
products is indicated. Numbers on abscissa are enzyme
units.
Figure 2: Expression and activity of truncated DRADA forms. A series of N- and C-terminally truncated DRADA versions is depicted on the left. The domain map of DRADA shows the three dsRBDs (boxed), the deaminase domain (arrowhead), the N- and C-terminal tags (arrows). The activity (mean ± S.D.) of the different DRADA forms on extended dsRNA (measured in nuclear extracts of transfected 293 cells) and for converting the adenosine of the GluR-B(R/G) site as assayed by primer extension on RT-PCR products from co-transfected cells is given for each construct with the number of experiments in parentheses. The expression of the different DRADA forms is documented by Western blot analysis with an anti-FLAG antibody.
We determined by DNA sequencing of cloned RT-PCR products from the in vitro editing reactions with both DRADA forms whether the adenosine deamination catalyzed by the recombinant enzymes was site-selective or promiscuous. As a result (not shown), adenosine conversion in the stem-loop RNA structure for the R/G site (6) was limited to the correct position, indicating positional fidelity by DRADA. In GluR6 pre-mRNA, the Q/R site adenosine and additional intronic positions, which are also edited in vivo, were found to be modified(9) . The analysis of products derived from pre-mRNA for the GluR-B Q/R site revealed that both DRADA versions had converted several adenosines other than that of the Q/R site, including positions -3, +3, +4, and 60 (hotspot1), but not the adenosine in the Q/R site itself (position 0). Collectively, our results indicate that recombinant DRADA can catalyze site-selective adenosine deamination in GluR pre-mRNAs and that at some sites this selectivity resembles that seen in vivo.
Figure 3:
DRADA-mediated editing of individual dsRNA
wild type and sequence-modified structures for different editing sites
in GluR pre-mRNAs. The dsRNAs tested as substrates for recombinant
DRADA are the wild type (bold) and sequence-modified GluR
pre-mRNAs for the Q/R site in GluR-B and GluR6, the GluR-B intron 11
hotspot1, and the GluR-B R/G site. The editing-targeted adenosine in
the predicted dsRNA structures(6, 8, 9) is
indicated in bold. In the mutated sites, sequence changes to
the wild type structure are boxed. Minigenes for the pre-mRNAs
were co-transfected into HEK 293 cells with or without a vector for
recombinant DRADA, or pre-mRNAs were in vitro synthesized and
incubated with purified recombinant DRADA. RT-PCR products were
analyzed for RNA editing by primer extension. For each editing site
tested, bar graphs (upper bars, cellular editing; lower bars, shaded, in vitro editing by 10 units of
recombinant N5 DRADA, see Fig. 1) indicate mean values of
adenosine conversion, line extensions to bars give
standard deviations, and the number of independent determinations is
listed in parentheses. For cellular editing, values from
co-transfection with DRADA are depicted by open parts of bars,
control values (no DRADA vector) are indicated by filled
parts. These cellular control values (only mean values are given)
were lower for GluR6 constructs (
0.4% for wt, M10) than
for GluR-B constructs (3-9%). Co-transfection data for GluR6 wt,
M10 and M11, are from (9) .
Figure 4: Gel filtration chromatography of DRADA and activities for GluR-B pre-mRNA editing. A, elution profile of the HiLoad 16/60 Superdex 200 gel filtration column. The editing of three sites (Q/R, R/G, hotspot1) in GluR-B pre-mRNA was determined for selected fractions. Arrows indicate the position and molecular masses of the marker proteins aldolase (158 kDa), bovine serum albumin (67 kDa), and ovalbumin (43 kDa). B, activity profile of DRADA in column fractions as assayed with dsRNA(22) . C, immunoblot with anti-DRADA (dsRBD) serum (1:4,000) ((17) ) of even-numbered column fractions 54-70, detected by chemiluminescence. Size markers (kilodaltons) are indicated on the right.
Western blotting of selected column fractions containing the Q/R site editing activity with antiserum against the first dsRBD of bovine DRADA (17) failed to detect DRADA. Moreover, the antiserum did not reveal a band corresponding in size to the Q/R site editing activity, which is smaller than DRADA (Fig. 4C). The <10% activity of adenosine conversion in extended dsRNA observed in the fraction with peak Q/R site editing activity compared with the DRADA peak fraction might derive from the Q/R site deaminase itself or from residual DRADA. However, at no point during purification could the Q/R site editing activity be enhanced by other fractions, including by peak fractions of DRADA (not shown). This appears to preclude the possibility that a factor from HeLa cells interacts with DRADA to generate Q/R site editing, as recently claimed for 293 cells(29) . Collectively, these data provide suggestive evidence for the existence in HeLa cells of an enzyme different from DRADA, possibly the recently cloned RED1 deaminase(30) , which can edit the Q/R site in GluR-B pre-mRNA.
DRADA is a candidate enzyme for mammalian nuclear transcript editing by adenosine deamination(2, 8, 14) . In the absence of characterized natural substrates for DRADA, the enzyme's activity has been primarily characterized with artificial extended dsRNAs in which DRADA can deaminate up to 50% of the adenosines(2, 14) . We tested the recombinant enzyme's activity at different editing positions in synthetic GluR transcripts and observed that DRADA edited to >90% the naturally mismatched adenosine of the GluR-B R/G site and the adenosine of hotspot1 in GluR-B intron 11, located in an A-U-rich environment next to a one-nucleotide bulge. The extent of DRADA-mediated editing at the GluR6 Q/R site with the adenosine positioned in an internal loop was lower, but this may reflect, in part, that the RNA tested for this site lacked a large segment of the native intron, potentially leading to inefficient RNA folding(9) .
Importantly, the Q/R site in GluR-B pre-mRNAs was not edited, and thus, contrary to recent speculations(2, 8, 16) , DRADA appears not to be involved in the editing of the GluR-B Q/R site. Fractionation of HeLa cell extracts suggests the existence of an editing activity distinct from DRADA, which can be separated from this enzyme by column chromatography, as reported by Yang et al.(12) . As shown here, this activity converts the adenosine of the GluR-B Q/R site, but not the adenosine of hotspot1 on the same substrate RNA(8) . Moreover, as predicted by the activity of recombinant DRADA on different GluR editing substrates, column fractions enriched in HeLa cell DRADA edited the R/G site and hotspot1, but not the GluR-B Q/R site, further substantiating the notion that these sites may serve as native substrates for DRADA. By testing truncated DRADA forms we largely excluded the possibility that the Q/R site editing activity of HeLa cells constitutes a smaller form of DRADA, generated by posttranslational or posttranscriptional processing. Additional differences between DRADA and the GluR-B Q/R site editing activity include the lack of cross-reactivity with a DRADA-specific anti-dsRBD serum (17) and the much smaller apparent size of the Q/R site editing activity, which would preclude the possibility that this activity represents DRADA complexed with a cellular factor. Therefore, the simplest explanation is that HeLa cells express a dsRNA-specific adenosine deaminase with distinct substrate specificity from DRADA.
A major determinant for the substrate selectivity by DRADA appears to be the local structure of the targeted adenosine, as revealed by mutational analysis. We observed that the Q/R site in GluR6 was not edited when the targeted adenosine was base paired (mutant M10), in analogy to the Q/R site of GluR-B. However, DRADA converted the GluR-B Q/R site adenosine at good efficiency when placed in an A-C mismatch configuration. These results are compatible with the view that DRADA can deaminate in vivo adenosines occupying mismatched positions in dsRNAs for the R/G site of AMPA receptor subunits GluR-B, -C, and -D and, possibly, the Q/R site in GluR5 and GluR6. Furthermore, DRADA may edit intronic hotspot1 in GluR-B pre-mRNA. Given the near ubiquitous expression of DRADA(31) , the enzyme is likely to edit pre-mRNAs in addition to those encoding GluR subunits. While such genes need to be characterized, the adenosine deamination in the intramolecular TAR stem-loop structure (32) may be generated by DRADA.
Notably, all sites putatively targeted by DRADA remain
largely unedited in the embryonic brain. During postnatal stages, these
sites, including the GluR-B intron 11 hotspot1 (embryonic day 14,
20% edited; postnatal day 0,
40%; P7,
50%; P14,
60%; P21 and P42,
70%), undergo a comparable developmental
progression in editing to an extent of 50-90% in the adult
brain(6, 33, 34) . Although DRADA expression
in brain appears to increase during brain development(17) ,
other gene products (35, 36) may also contribute to
such progressive editing. However, editing at these sites is
substantially below the >99% extent characteristic of the GluR-B Q/R
site. The almost complete adenosine conversion at this position is
essential for the low Ca
permeability of AMPA
receptors (37, 38) and the physiology of the central
nervous system(39) . Conversely, the change in kinetic
characteristics of AMPA receptor channels generated as a consequence of
R/G site editing (6) may play a role in the developmentally
regulated fine tuning of fast excitatory neurotransmission in central
synapses. Hence, different RNA editing enzymes appear to participate in
controlling the Ca
permeability and kinetic
properties of AMPA receptor channels.
Based on the present study, we interpret the pattern of adenosine deamination in exonic and intronic GluR-B pre-mRNA sequences from brain (8, 11) as reflecting the combined activity of different editing enzymes. DRADA preferentially converts adenosines in mismatched positions, loops, and bulges, probably because the altered geometry of the RNA helix (28) permits access by this enzyme. The GluR-B Q/R site adenosine appears to be deaminated by a different activity, possibly RED1(30) , which is molecularly related to DRADA.