Beyond the Identification of Transcribed Sequences:
Functional, Evolutionary and Expression Analysis
12th International Workshop
October 25-28, 2002
Washington, DC


List of Abstracts * Speakers * Organizers * Authors * Original Announcement


Intracellular Trafficking of A Few Inflammation-Inducible ADAR1 Isoforms

Jing-HuaYang, Qingchuan Zhao1, Yongzhan Nie, Yingjun Su, Koroush Kibir and Reuven Rabinovici
Department of Surgery, Yale University School of Medicine, New Haven, CT 06520
Email: jinghua.yang@yale.edu

ADAR1 is an editing enzyme catalyzing RNA-specific adenosine deamination in pre-mRNA. We have previously demonstrated that ADAR1 activity is induced in inflammatory cells during acute inflammation. Here we report that variable ADAR1 isoforms are produced and differentially localized in inflammatory and splenic cells. In in vitro activated splenocytes, intracellular localization of ADAR1 variants is shifted from the nucleus to the nucleolus and cytoplasm. This intracellular shift is proved due to the production of a few ADAR1 isoforms that lack nuclear localization signal, RNA recognition domains and/or Z-DNA binding domain through inflammation-inducible alternative splicing in exons 2 and 7, which are verified at both mRNA and protein levels. Studies in various cell types transfected with these ADAR1 variants demonstrate that the full-length ADAR1 and short variants deleting the C-terminals are localized in the cytoplasm. In contrast, short ADAR1 isoforms lacking the N-terminal sequences are detected in the nucleolus. Thus, ADAR1-mediated RNA editing is controlled at three different levels including inflammation-inducible transcription and alternative splicing as well as intracellular trafficking of ADAR1. It is indicated that A-to-I RNA editing may occur in the nucleus as well as the nucleolus and cytoplasm.


Introduction

A-to-I RNA editing is catalyzed by RNA-specific adenosine deaminase (ADAR) that converts adenosine to inosine and leads to the production of mRNA variants and protein isoforms. This process is ubiquitous and widely conserved as it was identified in multiple species including mammals (3, 14, 23, 26, 27), Xenopus(1), Drosophilae(28) and Zebrafish (37). To date, four A-to-I RNA editing enzymes, termed ADAR1, ADAR2, ADAR3 and ADAT1, have been cloned from mammals (3, 14, 23, 26, 27). ADAR1 and ADAR2 are widely expressed in a variety of cells and tissues (14, 27) with the highest expression in the brain and spleen. ADAR 3 was identified solely in the brain and its deaminase activity has not yet been established. ADAT1 targets tRNA and has been cloned from the human (23), mouse (24) and yeast (9).

ADARs are conserved in their adenosine deaminase domain but differ in their RNA binding domains. ADAR1 and ADAR2 contain two or three dsRNA binding domains (dsRBD) in addition to the adenosine deaminase domain. These two enzymes are capable of both non-specific editing of dsRNA and site-specific editing of glutamate receptor sub-unit B (gluR-B) pre-mRNA and serotonin receptor pre-mRNA (2, 40). ADAR2 selectively edits gluR-B at the Q/R site and serotonin at the D site whereas ADAR1 preferably targets gluR-B at the hot spot and serotonin at the A and C sites (2). Q/R site editing requires the formation of a base pairing structure around the editing site for specific substrate recognition by ADAR2.  The importance of the secondary structure in substrate recognition was confirmed in a study in which deletion of the stem loops around the Q/R editing site abolished the site-specific editing (40). ADAR3 and ADAT1 do not seem to edit these substrates.

A nuclear localization signal (NLS) and a Z-DNA binding domain are present near the N-terminal region of ADAR1 and are conserved in all species. This signal has also been identified as a nuclear export signal (NES) (32).  In addition, the human ADAR1 has an atypical NLS within its dsRBDIII and thus displays the characteristics of a shuttling protein (5). In contrast, the Xenopus ADAR1 contains a different NLS, which leads this enzyme to the nascent ribonucleoprotein matrix on the Xenopus lampbrush chromosomes and is specifically associated with active transcriptional sites. These findings suggest that the editing activity of Xenopus ADAR1 is coupled with transcriptional events or that the Xenopus ADAR1 targets newly synthesized RNAs (4).

Functional consequences of A-to-I RNA editing have been observed in the central nervous system. In the mammalian brain, editing by ADAR2 of gluR-B pre-mRNA has been shown to alter calcium permeability of excitatory neurons (6, 21). The role of ARAR2 was further studied in mice homozygous for a targeted functional null allele. In ADAR2-/- mice, A-to-I RNA editing was substantially reduced in diverse mRNAs and was coupled with seizure activity and early death (12). In Drosophila’s brain, disruption of the dADAR gene (a homologue of ADAR2) abolished sodium (Para), calcium (Dmca1A), and chloride (DrosGluCl- alpha) channels (10, 22, 29). Mutants lacking dADAR exhibited extreme behavioral deficits and neurodegeneration (29). Furthermore, a dADAR mutant displayed prolonged recovery from anoxic stupor, vulnerability to heat shock, and increased O2 demands (22). Thus, editing of ion channel pre-mRNAs by dADAR appears to be critical for the integrity and function of the central nervous system.

Several studies suggest that A-to-I RNA editing also plays a role in immune system. First, ADAR1 can be induced by interferon (IFN) in human amnion-U cells (31, 39) and pulmonary macrophages (33). Second, ADAR1 and ADAR2 can destroy dsRNA that may indirectly regulate the function of dsRNA binding proteins including IFN-induced PKR and 2', 5'-oligo(A) nuclease (31, 36). Third, studies in ADAR1 chimeric mouse embryos demonstrated that this editing enzyme affects embryonic erythropoiesis (38), suggesting that editing in this developmental stage is critical for normal erythrocyte proliferationand/or differentiation. We have recently reported that A-to-I RNA editing by ADAR1 is also involved in acute lung inflammation (33). Since acute inflammation is the underlying process of many critical illnesses including systemic inflammatory response syndrome (SIRS), multiple organ failure, sepsis, adult respiratory distress syndrome (ARDS), and ischemia/reperfusion injury (15), additional insight into the regulation of ADAR1-mediated RNA editing during inflammation could shed light into the pathogenesis of these conditions.

The present study further elucidates regulation and intracellular localization of ADAR1 enzyme in inflamed and activated splenocytes with special emphasis on the generation of several ADAR1 isoforms through inflammation-induced alternative splicing. In particular, different localizations of endogenous ADAR1 and transiently expressed isoforms in the cytoplasm, nucleus and nucleolus are demonstrated.

Materials and Methods

Animal model of acute inflammation.The model used was described previously in detail (13). In brief, endotoxin (LPS, sigma) at 15 mg/kg (LD50) was injected into the peritoneal cavity of conscious adult (6 week-old, 25 g) male C57Bl/6 mice (Jackson Laboratories, Bar Harbor, Me). Tissues were harvested after anesthesia (pentobarbital, 30 mg/kg) at several time points and processed for analysis as described below. The Yale Animal Care and Use Committee approved all animal protocols. Typically, five mice were used for each time point or experiment (n=5) and the same tissues were mixed for analysis.

Preparation of normal, activated and inflamed splenocytes. Normal and inflamed splenocytes were prepared from fresh spleens of normal or endotoxin-challenged (typically 15 mg/kg of LPS, 24 hours) mice. Spleens were cut into slides and passed through a strainer (Falcon). Red blood cells were removed by adding ACK cell lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3) and splenocytes were washed with PBS and collected by centrifugation. Typically 5x107 splenocytes were obtained from each spleen. For activation, 2x107 normal splenocytes were cultured in RPMI 1604 media in the presence of 100 units/ml of IL-2 or 2.5 mg/ml of Con-A for 48 hours. The activated splenocytes were collected by centrifugation. 

RNA and protein isolation. TotalRNA and mRNA were isolated from tissues or splenocytes using Trizol (Trizol, Inc.) and OligoTex (Qiagen) for Northern blotting, RT-PCR and ADAR1 cloning. Total protein was isolated by homogenizing splenocytes in four volumes of editing buffer (20 mM Hepes, pH 7.9, 100 mM KCl, 5 mM EDTA, 0.5% NP-40, 10% glycerol). The lysate was sonicated for 30 seconds and centrifuged for 30 seconds at 4,000 g.  Protein concentration in the supernatant was determined using BioRad protein kit and adjusted to 10 mg/ml for Western blotting or editing analyses.

Northern blotting, RT-PCR and Western analysis. Equalamounts  (~2 mg) of mRNA were used for Northern blotting. ADAR1 was detected by hybridization of the blots with 32P labeled antisense probes (1305-1265 and 3004-2966, AF291050).  Membranes were hybridized at 65°C over night and the final wash was with 0.1xSSC at 55°C for 10 min. For RT-PCR, 2 mg of total RNA were used for reverse transcription primed with poly(dT)12-18. ADAR1 mRNA was determined by RT-PCR using primers that flank exons 6-8 (1975-2003 and 2436-2408, AF291050) or the entire coding region (1-24 and 3459-3435, AF291050). The relative expression of ADAR1 mRNA was estimated in comparison to GAPDH. For Western blotting, 60 mg of total protein from splenic cells were resolved on 10% SDS-PAGE and transferred on a PVDF membrane. The blots were detected using antibodies against different regions of mouse ADAR1. The N-terminal antibody was produced with a synthetic peptide derived from the N-terminal sequence of mouse ADAR1 (Santa Cruz). Three C-terminal antibodies from different resources were used in the study: 1) antibodies developed in our laboratory using a recombinant protein of mouse ADAR1 (2765-3459 of AF291050), 2) antibodies against the C-terminal sequence of human ADAR1 (gift from Brenda Bass, Utah University) and 3) antibodies derived from a synthetic peptide of the C-terminal sequence of mouse ADAR1 (Santa Cruz).

Cloning and sequencing of ADAR1 variants. Products of the full-length ADAR1 cDNA from normal or inflamed splenic cells were cloned into pCRII (Invitrogen). Diversity of individual ADAR1 cDNA clones was analyzed by E.coRI digestion. The junction sites of alternative splicing were mapped by sequencing and analyzed by sequence alignment. Four typical ADAR1 cDNAs with open reading frame, ADAR1Sa (AF291875), ADAR1Sb (AF291877), ADAR1La (AF291050) and ADAR1Lb (AF291876), were subcloned for further analysis.

In vitro translation and expression of ADAR1 isoforms in a baculovirus system. The cDNA of ADAR1Sa, ADAR1Sb, ADAR1La or ADAR1Lb in the pCRII was translated in vitro using TNT T7 quick translation system following the manufacturer’s protocol (Promega). For recombinant proteins, these variants were subcloned into pFastBac (Gibco) in-frame with a His taq at the N-terminals to generate recombinant ADAR1-Bacmids. After transfection of Bacmids into Sf9 insect cells, recombinant ADAR1 isoforms were expressed and proteins isolated using Nickel columns following the manufacturer’s protocol (Pharmacia).

RNA editing assay using dsRNA.  RNA editing activity was evaluated by measuring A-to-I conversion of synthetic dsRNA (41). In a typical dsRNA editing assay, 10 ml of cell extracts (100 mg total protein) were mixed with 32P-labeled dsRNA (~5x103 cpm) and incubated at 37ºC for 1 hour. The mixture was treated with an equal volume of a PK buffer (10 mM Tris.HCl pH 8.0, 300 mM NaCl, 0.2% SDS, 0.5 mg/ml proteinase K). The edited dsRNA was extracted with phenol, precipitated in ethanol, resuspended in 10 ml of RNase P1 mix (5 mM Tris.Cl pH 7.5, 10 mM NaCl, 1 unit of RNase P1), and incubated at 37ºC for 2 hours. The converted inosine was analyzed on thin layer chromatography (TLC) and visualized by autoradiography after being developed in saturated (NH4)2SO4-isopropanol (95:5) solution. The radioactivity of each spot was quantified by scintillation.

Localization analysis of ADAR1-EGFP chimera and immunofluorescence. A restriction enzyme site (BamH I) was added at both ends of ADAR1 cDNA (ADAR1Sa, ADAR1Sb, ADAR1La and ADAR1Lb) by PCR using the pCRII-ADAR1 plasmids as templates. The PCR product was cleaved with BamHI and directly subcloned into pEGFP-N1 vector (Clontech) in-framed with the N-terminal of EGFP. Editing activity of the transiently expressed ADAR1-EGFP chimeras were tested in 293 cells and shown positive in converting adenosine to inosine on synthetic dsRNA. Different cells, including mouse fibroblasts (3T3), neuroblastoma (N18), monocytes (RAW264.7), or human HeLa cells,were transfected in triplets (n=3)with ADAR1-EGFP plasmids and fluorescence observed under microscope 6 hours after transfection. For immunofluorescence, cells were fixed on glass slides coated with poly-L-lysine and the specific proteins were hybridized with antibodies against ADAR1 or nucleolin and positive signals detected with fluorescence labeled second antibodies as described in the manufacturer’s protocol (Santa Cruz).

Results

Differential localization of endogenous ADAR1 variants. As ADAR1 is upregulated during acute inflammation (33) and the enzymatic activity of ADAR1 is to modify RNA, it is very important to determine the intracellular localization of ADAR1, which will help us to identify the RNAs that are targeted for editing. We first examined the localization of endogenous ADAR1 in a few cell lines using immunoflorescence. Mouse monocytes (RAW 264.7) were grown and fixed on glass slide covers coated with poly-L-lysine. The expression of ADAR1 protein was detected with antibodies against the N-terminal or C-terminal end of ADAR1. The anti-N-terminal antibody was to identify ADAR1 variants with intact N-terminals and anti-C-terminal antibodies were used to detect ADAR1 isoforms with intact C-terminals. As shown in Figure 1A, positive signals were dominantly detected in the cytoplasm of RAW cells when anti-N-terminal antibodies were used. However, signals from nucleolus-like particles were observed when anti-C-terminal antibodies were applied. Since both antibodies should detect the full-length ADAR1 protein, these results indicated that the full-length ADAR1 and/or variants with intact N-terminals were localized in the cytoplasm, while only ADAR1 variants with intact C-terminals were condensed in the nucleolus-like particles.

Interestingly, localization of ADAR1 not only varied in different cell types, but also differentially regulated during in vitro activation of splenocytes. In naïve mouse splenocytes, dominant signals were detected in the nucleus (Fig. 1B). However, an intracellular localization shift was observed when splenocytes were activated with IL-2 in vitro. Positive ADAR1 signals were found in the cytoplasm as well as the nucleolus-like particles in roughly 10% of splenocytes after activation. These findings indicated that the localization of endogenous ADAR1 was shifted from the nucleus to the cytoplasm and nucleolus in the activated splenocytes, which may reflect the production and differential localization of ADAR1 variants in inflammatory or splenic cells.

Multiple ADAR1 variants are induced in mouse splenocytes in response to inflammatory stimulation. To examine the expression of all possible ADAR1 variants, total mRNA from different mouse organs was analyzed by Northern blotting. RNA probes were prepared to hybridize with ADAR1 in the region of dsRNA-binding or catalytic domains to reduce non-specific signals. Two ADAR1 transcripts of approximately 7 and 5 kb in length were detected in all tested tissues (Fig. 2A). The expression of ADAR1 mRNA varied in different tissues with the highest signal in the brain and spleen and lowest in the liver. The ratio between the short and long transcripts also varied in a tissue-specific manner. The highest value was observed in the spleen and lowest in the brain (Fig. 2A). Similar results were obtained when the full-length ADAR1 cDNA was used as a probe. Further experiment demonstrated that both transcripts were significantly increased in the inflamed splenocytes from the endotoxin-challenged mice (Fig. 2B).

In order to characterize these transcripts, the cDNA fragment coding for the interferon-inducible ADAR1 protein (7, 8) was amplified from normal or inflamed mouse splenic cells using RT-PCR. Consistent with Northern blotting, a long and a short fragments were obtained, measuring 3.4 kb (termed ADAR1L) and 2.0 kb (termed ADAR1S), respectively (Fig. 2C). The expression of ADAR1S and ADAR1L was significantly increased after inflammatory stimulation. Especially, the expression of ADAR1S,that was twenty-fold less than that of ADAR1L in normal tissues,rapidly reached the level of ADAR1L. Careful analysis could reveal slightly different sizes of ADAR1S or ADAR1L (Fig. 2C), indicating more variations might occur. To characterize these variants, RT-PCR products of ADAR1L from spleens of normal or endotoxin-stimulated mice were cloned and individual cDNAs were analyzed by restriction enzyme. Three fragments (I, II and III) were identified, each covering exons 1-2, 2-5 or 5-15 of ADAR1, respectively (Fig. 2D and Fig. 3). Insertion or deletion mutations were indicated in fragments II and III because their sizes were different. In normal mouse splenic cells, two major variations in fragment III were obtained in 14 clones (Fig. 2D), while in stimulated splenocytes, three in fragment II and five in fragment III were found to be different out of 16 clones. Thus, not only the quantity but also the diversity of ADAR1 mRNA was increased during endotoxin-stimulated acute inflammation.

ADAR1 mRNA variants are generated by inflammation-induced alternative splicing. ADAR1 mRNA variants were analyzed by cloning and sequencing. Alignment analysis revealed that all variants were identical at their 5’ or 3’ ends. The difference between ADAR1L and ADAR1S was due to alternative selection of the 3’ splice site of intron 1, which resulted in a deletion of the entire exon 2 composed of approximately 1.4 kb of ADAR1 mRNA (Fig. 3A, C). Variations in fragment III resulted from alternative splicing of exon 7 due to selective usage of a few cryptic 5’ splice sites (Fig. 3B and C). Three ADAR1 variants with slightly different sizes, termed a-, b- and c-forms, were generated (Fig. 3C). The b-form matched the previously identified mouse ADAR1 cDNA (33) and was the dominant form in normal splenocytes (9 out of 14 clones). The a-form, which added 78-bp in exon 7 due to a cryptic 5’ splice site within intron 7, occurred less frequently (3 out of 14 clones).  This variation was also seen in the human ADAR1, in which a similar insertion was found to alter the specificity of editing on glutamate and serotonin receptor pre-mRNAs (19). The c-form, skipping the entire exon 7, was rarely noticed in normal mice. Interestingly, these variations in a-, b- and c-forms might alter substrate recognition or binding specificity because they all occurred in the dsRNA-binding domain III (RBDIII). In one clone with variation in fragment II, alternative splicing skipped the entire exon 3 (Fig. 3C), resulting in a deletion in the RBDII. 

         Variations in the dsRBDIII are differentially regulated through inflammation-induced alternative splicing. To study the regulation of the a-, b- and c-forms, RT-PCR was performed to amplify the fragment covering only exons from 5 to 8 of ADAR1. As shown in Figure 4, a- and b-forms were inversely regulated in mouse splenic cells after endotoxin stimulation. The a-form was progressively induced and sustained at a high level during the entire experiment, while the b-form was dominant in normal mice and gradually diminished 6 hours after endotoxin stimulation. These observations were in agreement with the cloning studies in which only the b-form was identified in healthy mice and more a-forms were found in the inflamed splenocytes (Fig. 2C). The levels of the c-form were relatively low in normal mice but upregulated in splenocytes after inflammatory stimulation (Fig. 4A). Because the a- and b-forms differ in their RNA binding domains, ADAR1 isoforms varying in this region may affect site-specific recognition and change the spectrum of RNA for editing in splenocytes during acute inflammation.

For comparison, expression of the a-, b- and c-forms in mouse brain was also analyzed under the same condition. Unlike in the spleen, the a-form instead of b-form was the dominant one in the brain (Fig. 4B) and the expression of the a-form in the brain was not regulated in response to endotoxin stimulation. However, the c-form was still upregulated in the similar way as that in the spleen. Therefore, the a- and b-forms are expressed in a tissue-specific manner and their productions are inversely regulated in the spleen but not in the brain.  

         ADAR1 protein isoforms with deletions at both N- and C-terminals are identified in splenic cells.Antibodies against the N- or three different C- terminal regions of the protein were used to analyze ADAR1 expression at the protein level. Theoretically, the identified ADAR1L and ADAR1S mRNAs should generate approximately 150 and 80 kD proteins, respectively, based on their sequences. The 80 kD protein was predicted from the open reading frame of ADAR1S mRNA starting at position 1555 (AF291050). As expected, a 150 kD protein was detected in splenocyte extracts by Western blotting using the anti-N- and anti-C-terminal antibodies (Fig. 5A), indicating that it was the full-length ADAR1 or ADAR1L150. A similar 150 kD ADAR1 is also known in human tissues (30). Two small protein bands measuring 115 and 80 kD, termed ADAR1S115 and ADAR1S80 respectively, were detected only when the anti-C-terminal antibodies were used (Fig. 5A), suggesting that they are ADAR1 isoforms which lack the N-terminal sequences. Apparently, ADAR1S80 is the product predicted from ADAR1S mRNA (Fig. 5C) whereas ADAR1S115 is probably from undefined alternatively spliced mRNAs.

Because the ADAR1L mRNA can be induced and also alternatively spliced to produce ADAR1S (Fig. 3), the overall expression of ADAR1L150 might vary with different stimulations. Indeed, the expression of ADAR1L150 in cultured splenocytes was upregulated following IL-2 stimulation but downregulated after Con-A stimulation (Fig. 5B). The expression of ADAR1S115 and ADAR1S80 was upregulated with all tested stimuli. In consistent with Northern and RT-PCR results, these findings indicated that the expression of the long and short ADAR1 isoforms were induced and differentially regulated in splenocytes after activation or inflammatory stimulation. Comparing with ADAR1L150, ADAR1S80 lacks the N-terminal NLS, the Z-DNA binding domain (11) and dsRBDI whereas ADAR1S115 probably lacks NLS and Za. Thus, it is conceivable that the function of ADAR1S80 and ADAR1S115 isoforms will be different from that of ADAR1L150.

Please note that slightly slower migrating protein corresponding to ADAR1S115 or ADAR1S80 was detected by Western blotting using antibodies against the C-terminal of ADAR1 (Fig. 5A, B). This protein was likely the modified variant of ADAR1S115 (or ADAR1S80) because it was also inducible after longer period of stimulation (unpublished data). In addition, two proteins with p90 and p100 kD were detected only when the N-terminal antibody was used, suggesting deletions also occur near the C-terminal end of ADAR1. Although these variants are likely inactive for RNA editing because of deletions in the catalytic domain, they might be differently localized and still be able to compete RNA substrates because of their intact RNA/Z-DNA binding domains and localization signals.

         Inflammation-inducible ADAR1 isoforms are functionally active in vitro. To test whether inflammation-inducible ADAR1L and ADAR1S80 isoforms with differentially regulated a- and b-forms (termed La, Lb, Sa and Sb, Fig. 6A) were functionally active, editing activity of these protein products was evaluated. La, Lb, Sa and Sb identical in their deaminase domain were translated in vitro and the sizes of their protein products were determined to correlate with the predicted sequences (Fig. 6B). To obtain sufficient quantities, the La, Lb, Sa and Sb proteins were produced in insect cells using a baculovirus expression system. The recombinant proteins containing a 6xhis tag at their N-terminals were purified and tested for editing of 32P-adensoine labeled dsRNA. As predicted, all isoforms were active to edit dsRNA and the activity varied with the highest in La and decreased in the order of Lb, Sa, and Sb (Fig. 6C).

Transiently expressed long and short ADAR1 isoforms are differentially localized in the cytoplasm and nucleolus.As the variations between ADAR1L150 and ADAR1S80 include the presence or absence of NLS and Z-DNA binding domain, these isoforms may be differentially localized in cells. To test this notion, La, Lb, Sa and Sb isoforms fused with EGFP at their C-terminals were first transfected into 3T3 cells and editing activity on dsRNA was examined. Results showed that editing activity was maintained in all chimeras (unpublished data) in spite of a previous report that mutations or deletions near the C-terminal of human ADAR1 affect RNA editing activity (16). Next, the localization of different ADAR1-EGFP chimeras was determined in transfected cells by fluorescence microscopy. The long ADAR1 variants were found in the cytoplasm (Fig. 7La, Lb), consistent with the human ADAR1 (32).

In contrast, ADAR1S80 isoforms were not only localized in the cytoplasm, but also accumulated as several bright particles in the nucleus (Fig. 7Sa, Sb). As these particles vary in size and number, ADAR1S80 appeared to reside in the nucleolus. This observation was confirmed by multi-color immunofluorescence study (Fig. 8). In this study, 3T3 cells transfected with ADAR1S80a-EGFP were fixed on glass slides and hybridized with antibodies against the nucleolar protein nucleolin (17). Visualization by fluorescence microscopy demonstrated co-localization of ADAR1-EGFP (green) and nucleolin (red), confirming the nucleolar localization of the ADAR1S80 isoforms. Considering the overall upregulation of ADAR1L150 and ADAR1S80 isoforms (Fig. 2 and 5), this result is in agreement with the increased cytoplasmic ADAR1 signal in the activated splenocytes (Fig. 1B). The accumulation of signals in the nucleolus (Fig. 1B) reflects the nucleolar binding capability of ADAR1S80 isoforms after stimulation.

The cytoplasmic localization ofADAR1L150 and nucleolar localization ADAR1S80aisoforms were examined in several cell types including mouse inflammatory cells (RAW 264.7), neuronal cell (N18) and human HeLa cell (Fig. 7B). In all cell types, ADAR1L150 was found in the cytoplasm and ADAR1S80a was distributed in the nucleolus and cytoplasm. Thus, the differential localization of ADAR1 isoforms occurred not only in fibroblasts but also in neuronal and inflammatory cells derived from the mouse and human. Further more, distribution pattern between the cytoplasm and nucleolus appeared to vary with cell types. In HeLa cells (Fig. 7B) ADAR1S80a was dominantly localized in the nucleolus, while in neuronal cells or fibroblasts significant cytoplasmic signal was observed.

Discussion

The present study demonstrates that that the localization of ADAR1 in mouse splenic cells is shifted from the nucleus to the nucleolus and cytoplasm after activation. This intracellular shift is proved due to the production of a few ADAR1 variants that lack nuclear localization signal, RNA recognition domains and/or Z-DNA binding domain.  In vitro studies further support that these functional ADAR1 variants are differentially localized in the nucleolus or the cytoplasm of all tested cell types. Differential localization of transiently expressed or endogenous ADAR1 isoforms delineates a novel mechanism to regulate RNA editing through coupling of inflammation-inducible transcription and alternative splicing and intracellular trafficking. Several important points are emphasized:

Production of multiple ADAR1 isoforms with distinct functional domains. The demonstration that diverse ADAR1 variants with distinct functional domains are produced in the inflamed splenocytes is a key finding of this study. More than a dozen ADAR1 mRNA variants, classified as ADAR1L or ADAR1S based upon their sizes, were found in the splenocytes from endotoxin-stimulated mice. The ADAR1S variants are produced by alternative splicing that deletes the entire exon 2 encoding the Z-DNA binding domain and NLS. At the protein level, a full-length ADAR1L150 and a few short isoforms including ADAR1S115 and ADAR1S80 were detected by Western blotting. The identified ADAR1L150 is comparable to the 150 kD human ADAR1, the product of the full length mRNA transcribed under the control of an interferon-inducible promoter (7). The mouse ADAR1S80 is likely the product of alternatively spliced ADAR1 mRNA that skips the entire exon 2 and deletes the NLS, the entire Z-DNA binding domain and the RBDI. This isoform was not only detected in mouse splenocytes, thymocytes and other mouse cell lines, but also found in human HeLa and gastric cancer cells (KATO-3) on Western blots (unpublished data), suggesting that ADAR1S80 is commonly expressed in mammals. However, the ADAR1S115 isoform may be comparable to the human 110-kD protein (30) that originates from a methionine 246 and retains the second half of the Z-DNA motif, all three dsRBDs and the catalytic domain. This variant is constitutively expressed and localized in the nucleus. The cDNA of mouse ADAR1S115 has not yet been cloned. It is either a product of different alternatively spliced mRNAs or a transcript from different promoters (7, 8).

The slower migrating bands near ADARS115 or ADARS80 proteins on Western blot are likely the different forms of ADARS115 or ADARS80 that are generated by post-translational modifications or alternative splicing. The present data prefers to the latter because the minor variation in the dsRBDIII will slightly change the size of ADARS115 or ADARS80. From sequence analysis, the size variation in this region will not only affect the function of RBDIII, but also change the distance from the catalytic domain to the N-terminal functional motifs. Further investigation needs to elucidate how the variations in this region contribute to the properties of ADARS115 or ADARS80 isoforms.

Differential regulation of ADAR1 isoforms in inflamed and activated splenocytes. Another key finding from this study is that ADAR1 isoforms are differentially regulated in the inflamed and activated splenocytes, which may provide a secondary control of ADAR1-mediated RNA editing. Results from RT-PCR, Northern and Western blotting show that ADAR1S115 and ADAR1S80 isoforms are induced in the activated splenocytes. However, ADAR1L150 expression was upregulated in IL-2 activated splenocytes but downregulated in the same cells after Con-A activation. Its overall expression in the inflamed splenocytes will depend on transcription and alternative splicing of ADAR1 transcripts. In addition to it, there is a preferential production of the a-form ADAR1 with increasing amount of c-forms. In contrast, the dominant b-forms in normal animals are quickly downregulated after inflammatory stimulation. Consequently, this differential regulation of ADAR1 isoforms during splenocyte activation will result in an increase of ADAR1L150a, ADAR1S80a, ADAR1L150c and ADAR1S80c and a decrease of ADAR1S150b and ADAR1S80b isoforms. Previous studies demonstrate that deletion of dsRNA binding I, II or III affects the editing activity as well as the specificity of ADAR1 enzyme (25). A similar alternative splicing in the dsRBDIII of human ADAR1 affects the site specificity of serotonin receptor pre-mRNA editing (18-20, 41). Therefore, these differently regulated ADAR1 isoforms in splenocytes are conceivably to target different spectrum of mRNAs for editing and lead to functional consequences on splenic cells.

The mechanisms responsible for the differential regulation of ADAR1 isoforms are not yet clear. Nevertheless, alternative splicing factors that are required to enhance the skipping of the entire exon 2 and/or activate the multiple 5’ splice sites of exon 7 may be induced during inflammation. The a- and b-forms appear to be generated through competitive selection of two different 5’ splice sites of intron 7. This is supported by the reciprocal response of a- and b-forms to inflammatory stimulation.  Since the induction of the c-form appears to be less significant, mechanisms other than competition should be contemplated. For example, it is possible that the generation of the c- form is secondary to the induction of independent splicing factors or to cell specific alternative splicing. Another potential regulatory mechanism of ADAR1 isoform production could be autoregulation by ADAR1 itself. The possibility that ADAR1 regulates the diversity of its own gene products is supported by previous reports that ADARs are capable of editing their own mRNA (35), interacting with spliceosomes (34), and regulating pre-mRNA splicing (2). Thus, the coupling of alternative splicing and inflammation-induced RNA editing could be one unique feature of the ADAR editing enzymes.

Regulation of intracellular trafficking of ADAR1 isoforms. The most important finding from this study is that the inflammation-regulated ADAR1 isoforms are differentially localized in the cytoplasm and the nucleolus in the activated splenocytes, which could reflect an additional level of regulatory mechanism for ADAR1-mediated RNA editing. For instance, it may differentially channel the induced ADAR1 isoforms in splenocytes to specific intracellular sites during immune or inflammatory responses.  

It is demonstrated in a variety of cell types that ADAR1L150 is accumulated in the cytoplasm whereas ADAR1S80is localized in the cytoplasm as well as the nucleolus. The cytoplasmic localization of ADAR1L150 is in agreement with that of human ADAR1 (32), suggesting that this phenomenon could be consistent in mammals. The nucleolar binding capability of ADAR1S80 isoforms can be explained by the presence of a typical basic-residue motif for nucleolar localization near the dsRBDIII (unpublished data). This motif also exists in the human ADAR1 and overlaps with the nucleus import signal that was recently mapped in the dsRBDIII (5). Markedly, localization of the endogenous ADAR1 in splenocytes is different before and after the activation with IL-2. In the activated splenocytes, endogenous ADAR1 proteins dominantly reside in the cytoplasm and nucleolus whereas in the naïve cells ADAR1 signals are detected in the nucleus. Thus, an intracellular localization shift of endogenous ADAR1 from the nucleus to cytoplasm and nucleolus is demonstrated in response to IL-2 stimulation. The upregulation of ADAR1L150 and ADAR1S80 in IL-2 activated splenocytes supports the increased ADAR1 signal in the cytoplasm and nucleolus. The nuclear signal in the naive splenocytes could be caused by ADAR1S115 because the comparable human 110 kD ADAR1 is localized in the nucleus (30). However, it still remains unclear how the localization of the N-terminal ADAR1 fragments contributes to ADAR1 signals in the nucleus of naive splenocytes. Investigation is underway to clone the cDNA of ADAR1S115 and characterize its expression and localization regulation.

Identification of ADAR1 isoforms in the cytoplasm, nucleus and nucleolus indicates that A-to-I RNA editing could occur in different intracellular sites. Our observation that the short ADAR1 isoforms are localized in the nucleolus indicates possible occurrence of ADAR1-mediated RNA editing on nucleolar RNAs (i.e. pre-rRNA, snoRNAs), which has not yet been documented. The cytoplasmic localization of full-length ADAR1 isoforms reveals a potential editing of cytoplasmic RNA (i.e. mature mRNA, rRNA or viral RNAs). In contrast, RNA editing in the nucleus could be inferred from several previous observations. For instance, RNA editing by ADAR1 and ADAR2 of gluR-B and serotonin pre-mRNA requires intron sequences (2, 27, 40), suggesting the editing event is in the nucleus. Recently, the human but not the Xenopus ADAR1 has been shown to shuttle between cytoplasm and nucleus (5) and the Xenopus ADAR1 without NLS accumulated in the nucleus of oocytes (4). This Xenopus ADAR1 is localized in the nucleus that binds the nascent ribonucleoprotein matrix on lampbrush chromosomes and is specifically associated with transcriptionally active loops (4). Thus, the nucleus localization and its association with transcriptionally active sites are likely to facilitate editing of newly synthesized RNA precursors. Taken together, our findings indicated that during splenocyte activation or under inflammatory conditions the differentially regulated ADAR1 isoforms are capable of editing wider spectrum of RNAs in the nucleus as well as in the cytoplasm and nucleolus.

In summary, the present study sheds light into the role of inflammation-induced ADAR1 isoforms during splenocyte activation, which reveals a novel regulatory mechanism of ADAR1-mediated RNA editing through coupling of alternative spicing and intracellular localization during inflammatory or immune responses.

Acknowledgement

We thank Alfred Bothwell and Chou Hung for helpful comments.  Q. Zhao and Y. Nie contributed equally to this work.

This work was supported by NIH Grants GM-60426 to J.-H.Y. and HL57963-01to R.R. It is also partially supported by the Natural Science Foundation of China Grants 39870418, 39870176 and 30021002 and a Hellman Foundation Fellowship to J.-H.Y.

 

References


Figure Legends:

Fig. 1. Intracellular distribution of endogenous ADAR1 proteins. A. ADAR1 localization in mouse RAW 264.7 cells detected with antibodies against the N- or C-terminal of ADAR1, indicated as N-terminal-Ab and C-terminal-Ab, respectively. B.  ADAR1 localization in naïve or IL-2 activated splenocytes that were fixed on glass slide covers and stained with antibodies against the catalytic domain of ADAR1 protein. N, nucleus; No, nucleolus; C, cytoplasm. Bar, 5 mm.

Fig. 2. Diverse ADAR1 variants are produced and upregulated in immune organs after endotoxin stimulation. A. Northern blotting analysis. Two ADAR1 mRNAs are expressed in a tissue-specific manner in all tested mouse tissues. B.Northern blotting analysis. Two ADAR1 mRNAs are induced in spleens harvested 4 hours after endotoxin-stimulation (analysis by Northern blotting). C. RT-PCR analysis.Long (~3.4 kb, L) and short (~2.0 kb, S) ADAR1 variants are induced in spleens harvested 4 hours after endotoxin stimulation. RT-PCR was performed using primers covering the entire coding region. D. A variety of long ADAR1 isoforms are induced by inflammation. Individual cDNAs that were cloned from the spleens of sham (0h) and endotoxin stimulated (4h) mice were analyzed by EcoRI enzyme digestion. Please note that more variations in fragment II and III are detected in endotoxin-challenged tissues. v, pCRII vector. I, II and III, fragments I, II and III, respectively. b-actin and GAPDH, internal controls.

Fig. 3. Sequences and scheme of alternatively spliced ADAR1 variants. A. Sequence alignment of the long (ADAR1La: AF291050, ADAR1Lb: AF291876) and short (ADAR1Sa: AF291875, ADAR1Sb: AF291877) variants. Underlined, translation start codon; Dashed line, deleted region. B. Sequence alignment of the alternatively spliced forms a, b and c. Sequences are from ADAR1La (AF291050) and ADAR1Lb (AF291876).  C.  Schematic summary of alternative splicing in mouse ADAR1. L/S, alternative splicing that skips exon 2 and generates the long and short variants. a, b and c, alternative splicing that results in minor deletions or insertions in exon 7.  I, II and III indicate E.coRI digested fragments I, II and III, respectively.

Fig. 4. Differential regulation of inflammation-inducible ADAR1 mRNAs with variations in the dsRBDIII region. A and B. RT-PCR analysis of mouse ADAR1 fragment from exon 5 to 8. Three fragments of ADAR1, termed a-, b- and c-forms, are produced and differentially regulated in spleens (A) or brains (B) of mice that are challenged with endotoxin for 0, 2, 4, 6, 16 and 20 hours. Note that the a- and b-forms are regulated differently in spleen or brain whereas the c-form is induced in both tissues. GAPDH is an internal control. C. Scheme of alternative splicing that generates a-, b- and c-forms of ADAR1 in normal (top) or inflamed (bottom) splenic cells. Three different 5’ alternative splicing sites are indicated. The b-form is dominant under normal conditions, while the a-form becomes dominant and the c-form increases during endotoxin-induced inflammation.

Fig. 5. Production and differential regulation of the long and a few short ADAR1 protein isoforms in splenic cells. A. Western blotting analysis of the long (L) and a few short (S) ADAR1 proteins in cultured splenocytes. The full-length protein (150 kD) and a few small isoforms (~115 and 80 kD) lacking variable length at their N-terminals were detected with antibodies against the C-terminal of ADAR1 (C-term). Two fragments (100 and 90 kD) missing partial deaminase domain were detected with antibody against the N-terminal of ADAR1 (C-term). B. Differential expression of the full-length ADAR1 and the short isoforms in naïve (N) and in IL-2 or Con-A activated (48 hours in vitro) splenic cells.  Proteins were detected with antibodies against the C-terminal of ADAR1. C. Scheme for alternative splicing of ADAR1L150 and ADAR1S80 isoforms in normal (bottom) or activated (top) splenic cells. Bold bar, RT-PCR primer. 

Fig. 6. Editing activity of four ADAR1 isoforms. A. Scheme of ADAR1L150 and ADAR1S80 variants with a- or b-form in dsRBDIII, termed La, Lb, Sa and Sb, respectively. Stripped area, Z-DNA binding domain; shadowed area, dsRBDs; triangle, NLS or putative nucleolar binding signal; dots, the deaminase domain; open box, insert in dsRBDIII.  B. SDS-PAGE analysis of in vitro translated La, Lb, Sa and Sb.  Note that the size of all variants corresponds to their sequences. C. TLC analysis of dsRNA editing activity of the recombinant La, Sa, Lb and Sb expressed in Sf9 cells using baculovirus system. The ratio between pI (5’-monophosphate inosine) and pA (5’-monophosphate adenosine) represents A-to-I editing activity.

Fig 7. Cytoplasmic and nucleolar localization of transiently expressed ADAR1L150 and ADAR1S80. A. Localization of ADAR1L150 and ADAR1S80 with a and b variations. La, Lb, Sa and Sb variants were tagged with EGFP at the C-terminals and tested positive for dsRNA editing by transient transfection of fibroblasts. Localization of ADAR1-EGFP chimeras was then analyzed by transient transfection of the same cells and visualized by fluorescence microscopy. The long variants, La and Lb, were found in the cytoplasm. The short variants, Sa and Sb, were found in the nucleus and formed irregular sharp multiple nucleolus-like particles. B. Cytoplasmic or nucleolar localization of ADAR1 isoforms in various cell types. Transiently expressed of ADAR1La and ADAR1S80a was examined in neuronal (N18), monocytic (RAW 264.7) and HeLa cells and visualized by fluorescence microscopy. Note that ADAR1L150 and ADAR1S80a were localized in the cytoplasm and nucleolus, respectively. Bar, 10 mm. 

Fig. 8. Nucleolar localization of transiently expressed ADAR1S80a. HeLa cells transfected with the short ADAR1S80a-EGFP were detected by fluorescence microscopy (A, green) and immunofluorescence using antibodies against the nucleolar protein nucleolin (C23) and TRITC-conjugated second antibodies (B, red). ADAR1 (green) and nucleolin (red) were digitalized and superimposed (C), confirming the nucleolar localization of ADAR1S80a. Bar, 5 mm.

1.   Bass, B. L., and H. Weintraub 1988. An unwinding activity that covalently modifies its double-stranded RNA substrate Cell. 55:1089-98.

2.   Burns, C. M., H. Chu, S. M. Rueter, L. K. Hutchinson, H. Canton, E. Sanders-Bush, and R. B. Emeson 1997. Regulation of serotonin-2C receptor G-protein coupling by RNA editing [see comments] Nature. 387:303-8.

3.   Chen, C. X., D. S. Cho, Q. Wang, F. Lai, K. C. Carter, and K. Nishikura 2000. A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both single- and double-stranded RNA binding domains [In Process Citation] Rna. 6:755-67.

4.   Eckmann, C. R., and M. F. Jantsch 1999. The RNA-editing enzyme ADAR1 is localized to the nascent ribonucleoprotein matrix on Xenopus lampbrush chromosomes but specifically associates with an atypical loop J Cell Biol. 144:603-15.

5.   Eckmann, C. R., A. Neunteufl, L. Pfaffstetter, and M. F. Jantsch 2001. The human but not the Xenopus RNA-editing enzyme ADAR1 has an atypical nuclear localization signal and displays the characteristics of a shuttling protein Mol Biol Cell. 12:1911-24.

6.   Feldmeyer, D., K. Kask, R. Brusa, H. C. Kornau, R. Kolhekar, A. Rozov, N. Burnashev, V. Jensen, O. Hvalby, R. Sprengel, and P. H. Seeburg 1999. Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B Nat Neurosci. 2:57-64.

7.   George, C. X., and C. E. Samuel 1999. Characterization of the 5'-flanking region of the human RNA-specific adenosine deaminase ADAR1 gene and identification of an interferon- inducible ADAR1 promoter Gene. 229:203-13.

8.   George, C. X., and C. E. Samuel 1999. Human RNA-specific adenosine deaminase ADAR1 transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible Proc Natl Acad Sci U S A. 96:4621-6.

9.   Gerber, A., H. Grosjean, T. Melcher, and W. Keller 1998. Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2 Embo J. 17:4780-9.

10.Hanrahan, C. J., M. J. Palladino, B. Ganetzky, and R. A. Reenan 2000. RNA editing of the Drosophila para Na(+) channel transcript. Evolutionary conservation and developmental regulation Genetics. 155:1149-60.

11.Herbert, A., J. Alfken, Y. G. Kim, I. S. Mian, K. Nishikura, and A. Rich 1997. A Z-DNA binding domain present in the human editing enzyme, double- stranded RNA adenosine deaminase Proc Natl Acad Sci U S A. 94:8421-6.

12.Higuchi, M., S. Maas, F. N. Single, J. Hartner, A. Rozov, N. Burnashev, D. Feldmeyer, R. Sprengel, and P. H. Seeburg 2000. Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2 [In Process Citation] Nature. 406:78-81.

13.Kabir, K., J. P. Gelinas, M. Chen, D. Chen, D. Zhang, X. Luo, J. H. Yang, D. Carter, and R. Rabinovici 2002. Characterization of a murine model of endotoxin-induced acute lung injury Shock. 17:300-3.

14.Kim, U., Y. Wang, T. Sanford, Y. Zeng, and K. Nishikura 1994. Molecular cloning of cDNA for double-stranded RNA adenosine deaminase, a candidate enzyme for nuclear RNA editing Proc Natl Acad Sci U S A. 91:11457-61.

15.Krafft, P., P. Fridrich, T. Pernerstorfer, R. D. Fitzgerald, D. Koc, B. Schneider, A. F. Hammerle, and H. Steltzer 1996. The acute respiratory distress syndrome: definitions, severity and clinical outcome. An analysis of 101 clinical investigations Intensive Care Med. 22:519-29.

16.Lai, F., R. Drakas, and K. Nishikura 1995. Mutagenic analysis of double-stranded RNA adenosine deaminase, a candidate enzyme for RNA editing of glutamate-gated ion channel transcripts J Biol Chem. 270:17098-105.

17.Li, Y. P., R. K. Busch, B. C. Valdez, and H. Busch 1996. C23 interacts with B23, a putative nucleolar-localization-signal- binding protein Eur J Biochem. 237:153-8.

18.Liu, Y., R. B. Emeson, and C. E. Samuel 1999. Serotonin-2C receptor pre-mRNA editing in rat brain and in vitro by splice site variants of the interferon-inducible double-stranded RNA- specific adenosine deaminase ADAR1 J Biol Chem. 274:18351-8.

19.Liu, Y., C. X. George, J. B. Patterson, and C. E. Samuel 1997. Functionally distinct double-stranded RNA-binding domains associated with alternative splice site variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase J Biol Chem. 272:4419-28.

20.Liu, Y., and C. E. Samuel 1999. Editing of glutamate receptor subunit B pre-mRNA by splice-site variants of interferon-inducible double-stranded RNA-specific adenosine deaminase ADAR1 J Biol Chem. 274:5070-7.

21.Lomeli, H., J. Mosbacher, T. Melcher, T. Hoger, J. R. Geiger, T. Kuner, H. Monyer, M. Higuchi, A. Bach, and P. H. Seeburg 1994. Control of kinetic properties of AMPA receptor channels by nuclear RNA editing Science. 266:1709-13.

22.Ma, E., X. Q. Gu, X. Wu, T. Xu, and G. G. Haddad 2001. Mutation in pre-mRNA adenosine deaminase markedly attenuates neuronal tolerance to O2 deprivation in Drosophila melanogaster J Clin Invest. 107:685-93.

23.Maas, S., A. P. Gerber, and A. Rich 1999. Identification and characterization of a human tRNA-specific adenosine deaminase related to the ADAR family of pre-mRNA editing enzymes Proc Natl Acad Sci U S A. 96:8895-900.

24.Maas, S., Y. G. Kim, and A. Rich 2000. Sequence, genomic organization and functional expression of the murine tRNA-specific adenosine deaminase ADAT1 Gene. 243:59-66.

25.Maas, S., T. Melcher, A. Herb, P. H. Seeburg, W. Keller, S. Krause, M. Higuchi, and M. A. O'Connell 1996. Structural requirements for RNA editing in glutamate receptor pre-mRNAs by recombinant double-stranded RNA adenosine deaminase J Biol Chem. 271:12221-6.

26.Melcher, T., S. Maas, A. Herb, R. Sprengel, M. Higuchi, and P. H. Seeburg 1996. RED2, a brain-specific member of the RNA-specific adenosine deaminase family J Biol Chem. 271:31795-8.

27.Melcher, T., S. Maas, A. Herb, R. Sprengel, P. H. Seeburg, and M. Higuchi 1996. A mammalian RNA editing enzyme Nature. 379:460-4.

28.Palladino, M. J., L. P. Keegan, M. A. O'Connell, and R. A. Reenan 2000. dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing Rna. 6:1004-18.

29.Palladino, M. J., L. P. Keegan, M. A. O'Connell, and R. A. Reenan 2000. A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity Cell. 102:437-49.

30.Patterson, J. B., and C. E. Samuel 1995. Expression and regulation by interferon of a double-stranded-RNA- specific adenosine deaminase from human cells: evidence for two forms of the deaminase Mol Cell Biol. 15:5376-88.

31.Patterson, J. B., D. C. Thomis, S. L. Hans, and C. E. Samuel 1995. Mechanism of interferon action: double-stranded RNA-specific adenosine deaminase from human cells is inducible by alpha and gamma interferons Virology. 210:508-11.

32.Poulsen, H., J. Nilsson, C. K. Damgaard, J. Egebjerg, and J. Kjems 2001. CRM1 mediates the export of ADAR1 through a nuclear export signal within the Z-DNA binding domain Mol Cell Biol. 21:7862-71.

33.Rabinovici, R., K. Kabir, M. Chen, Y. Su, D. Zhang, X. Luo, and J. H. Yang 2001. ADAR1 is involved in the development of microvascular lung injury Circ Res. 88:1066-71.

34.Raitskin, O., D. S. Cho, J. Sperling, K. Nishikura, and R. Sperling 2001. RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery Proc Natl Acad Sci U S A. 98:6571-6.

35.Rueter, S. M., T. R. Dawson, and R. B. Emeson 1999. Regulation of alternative splicing by RNA editing Nature. 399:75-80.

36.Samuel, C. E. 1998. Reoviruses and the interferon system Curr Top Microbiol Immunol. 233:125-45.

37.Slavov, D., M. Clark, and K. Gardiner 2000. Comparative analysis of the RED1 and RED2 A-to-I RNA editing genes from mammals, pufferfish and zebrafish Gene. 250:41-51.

38.Wang, Q., J. Khillan, P. Gadue, and K. Nishikura 2000. Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis Science. 290:1765-8.

39.Weier, H. U., C. X. George, K. M. Greulich, and C. E. Samuel 1995. The interferon-inducible, double-stranded RNA-specific adenosine deaminase gene (DSRAD) maps to human chromosome 1q21.1-21.2 Genomics. 30:372-5.

40.Yang, J. H., P. Sklar, R. Axel, and T. Maniatis 1995. Editing of glutamate receptor subunit B pre-mRNA in vitro by site- specific deamination of adenosine Nature. 374:77-81.

41.Yang, J. H., P. Sklar, R. Axel, and T. Maniatis 1997. Purification and characterization of a human RNA adenosine deaminase for glutamate receptor B pre-mRNA editing Proc Natl Acad Sci U S A. 94:4354-9.



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