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Distinct in vivo roles for double-stranded RNA-binding domains of the Xenopus RNA-editing enzyme ADAR1 in chromosomal targeting.

Doyle M, Jantsch MF - J. Cell Biol. (2003)

Bottom Line: Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally.Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA-binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein.Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell Biology and Genetics, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria.

ABSTRACT
The RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) deaminates adenosines to inosines in double-stranded RNA substrates. Currently, it is not clear how the enzyme targets and discriminates different substrates in vivo. However, it has been shown that the deaminase domain plays an important role in distinguishing various adenosines within a given substrate RNA in vitro. Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally. Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA-binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein. Most importantly, we show that individual dsRBDs are capable of recognizing different chromosomal sites in an apparently specific manner. Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

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Individual dsRBDs can lead to ADAR1 enrichment on different transcription units. RNA transcribed from full-length myc-tagged ADAR1 constructs with either a single copy of dsRBD1 (A) or dsRBD2 (B) in place of the three endogenous dsRBDs was injected into oocytes. Translation of both constructs was followed using mAb 9E10 and a secondary FITC-labeled antibody. Simultaneously, all preparations were stained for endogenous ADAR1 using the SAT3 antisera (endogenous). Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Both dsRBD constructs were able to restore at least some level of chromosomal labeling, but at reduced levels compared with the endogenous ADAR1 staining. Interestingly, labeling often appeared at the same position along the arms of each homologue (A and B, arrows) indicating specific targeting to the same chromosomal loops. (C) To determine the influence of overall RNA–binding strength, constructs containing a duplication (dsRBD 2-2 GFP) or a triplication (dsRBD 2-2-2 MYC) of dsRBD were tagged with GFP and myc, respectively, injected, and detected within the same oocyte. Both constructs did label the same chromosomal sites (C, arrows), thus leading to a homogeneous yellow labeling in the overlay (merge). (D) To directly compare two individual dsRBDs on the same chromosome, RNA, transcribed from GFP-tagged ADAR1 containing a single dsRBD1, was coinjected with RNA made from a myc-tagged ADAR1 construct containing a single dsRBD2. Translation of the GFP-tagged construct was followed using appropriate antibodies. The dsRBD2-containing construct shows specific enrichment at a few chromosomal sites (D, top arrows), whereas the dsRBD1-containing construct shows a more homogeneous chromosomal staining. However, a few sites are specifically highlighted by the dsRBD1-containing constructs and, thus, appear green in the merged image (D, bottom arrows). Merged images of dsRBD1 and dsRBD2 labeling are shown (merge) as well as images of the chromosomes taken by differential interference contrast (NOM). Bar, 20 μm
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fig5: Individual dsRBDs can lead to ADAR1 enrichment on different transcription units. RNA transcribed from full-length myc-tagged ADAR1 constructs with either a single copy of dsRBD1 (A) or dsRBD2 (B) in place of the three endogenous dsRBDs was injected into oocytes. Translation of both constructs was followed using mAb 9E10 and a secondary FITC-labeled antibody. Simultaneously, all preparations were stained for endogenous ADAR1 using the SAT3 antisera (endogenous). Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Both dsRBD constructs were able to restore at least some level of chromosomal labeling, but at reduced levels compared with the endogenous ADAR1 staining. Interestingly, labeling often appeared at the same position along the arms of each homologue (A and B, arrows) indicating specific targeting to the same chromosomal loops. (C) To determine the influence of overall RNA–binding strength, constructs containing a duplication (dsRBD 2-2 GFP) or a triplication (dsRBD 2-2-2 MYC) of dsRBD were tagged with GFP and myc, respectively, injected, and detected within the same oocyte. Both constructs did label the same chromosomal sites (C, arrows), thus leading to a homogeneous yellow labeling in the overlay (merge). (D) To directly compare two individual dsRBDs on the same chromosome, RNA, transcribed from GFP-tagged ADAR1 containing a single dsRBD1, was coinjected with RNA made from a myc-tagged ADAR1 construct containing a single dsRBD2. Translation of the GFP-tagged construct was followed using appropriate antibodies. The dsRBD2-containing construct shows specific enrichment at a few chromosomal sites (D, top arrows), whereas the dsRBD1-containing construct shows a more homogeneous chromosomal staining. However, a few sites are specifically highlighted by the dsRBD1-containing constructs and, thus, appear green in the merged image (D, bottom arrows). Merged images of dsRBD1 and dsRBD2 labeling are shown (merge) as well as images of the chromosomes taken by differential interference contrast (NOM). Bar, 20 μm

Mentions: To assess the role of individual domains in chromosomal targeting, we initially compared the distribution of single domain constructs with that of the endogenous protein. In these experiments, no single domain was able to mimic endogenous staining including enrichment at the special loop. Interestingly, however, any individual domain was capable of restoring at least some level of chromosomal localization. Unlike the distribution of full-length ADAR1, constructs bearing dsRBD1 or dsRBD2 predominantly highlighted a few loops on all chromosomes (Fig. 5) , whereas the dsRBD3-containing construct showed a rather weak and relatively homogeneous staining (unpublished data). Moreover, chromosomal loops that were labeled by individual domains were always stained by the endogenous antibody, but not vice versa (Fig. 5). To ensure that the observed labeling was not due to insufficient incubation time, and thus, reduced translation product levels, oocytes were cultured for up to 10 d. Assessment at different time points showed that labeling increased slightly over the first 48-h period, but remained constant for the remainder of the experiment. Additionally, Western blots of injected oocytes were made to control for equal and sufficient protein production (unpublished data).


Distinct in vivo roles for double-stranded RNA-binding domains of the Xenopus RNA-editing enzyme ADAR1 in chromosomal targeting.

Doyle M, Jantsch MF - J. Cell Biol. (2003)

Individual dsRBDs can lead to ADAR1 enrichment on different transcription units. RNA transcribed from full-length myc-tagged ADAR1 constructs with either a single copy of dsRBD1 (A) or dsRBD2 (B) in place of the three endogenous dsRBDs was injected into oocytes. Translation of both constructs was followed using mAb 9E10 and a secondary FITC-labeled antibody. Simultaneously, all preparations were stained for endogenous ADAR1 using the SAT3 antisera (endogenous). Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Both dsRBD constructs were able to restore at least some level of chromosomal labeling, but at reduced levels compared with the endogenous ADAR1 staining. Interestingly, labeling often appeared at the same position along the arms of each homologue (A and B, arrows) indicating specific targeting to the same chromosomal loops. (C) To determine the influence of overall RNA–binding strength, constructs containing a duplication (dsRBD 2-2 GFP) or a triplication (dsRBD 2-2-2 MYC) of dsRBD were tagged with GFP and myc, respectively, injected, and detected within the same oocyte. Both constructs did label the same chromosomal sites (C, arrows), thus leading to a homogeneous yellow labeling in the overlay (merge). (D) To directly compare two individual dsRBDs on the same chromosome, RNA, transcribed from GFP-tagged ADAR1 containing a single dsRBD1, was coinjected with RNA made from a myc-tagged ADAR1 construct containing a single dsRBD2. Translation of the GFP-tagged construct was followed using appropriate antibodies. The dsRBD2-containing construct shows specific enrichment at a few chromosomal sites (D, top arrows), whereas the dsRBD1-containing construct shows a more homogeneous chromosomal staining. However, a few sites are specifically highlighted by the dsRBD1-containing constructs and, thus, appear green in the merged image (D, bottom arrows). Merged images of dsRBD1 and dsRBD2 labeling are shown (merge) as well as images of the chromosomes taken by differential interference contrast (NOM). Bar, 20 μm
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fig5: Individual dsRBDs can lead to ADAR1 enrichment on different transcription units. RNA transcribed from full-length myc-tagged ADAR1 constructs with either a single copy of dsRBD1 (A) or dsRBD2 (B) in place of the three endogenous dsRBDs was injected into oocytes. Translation of both constructs was followed using mAb 9E10 and a secondary FITC-labeled antibody. Simultaneously, all preparations were stained for endogenous ADAR1 using the SAT3 antisera (endogenous). Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Both dsRBD constructs were able to restore at least some level of chromosomal labeling, but at reduced levels compared with the endogenous ADAR1 staining. Interestingly, labeling often appeared at the same position along the arms of each homologue (A and B, arrows) indicating specific targeting to the same chromosomal loops. (C) To determine the influence of overall RNA–binding strength, constructs containing a duplication (dsRBD 2-2 GFP) or a triplication (dsRBD 2-2-2 MYC) of dsRBD were tagged with GFP and myc, respectively, injected, and detected within the same oocyte. Both constructs did label the same chromosomal sites (C, arrows), thus leading to a homogeneous yellow labeling in the overlay (merge). (D) To directly compare two individual dsRBDs on the same chromosome, RNA, transcribed from GFP-tagged ADAR1 containing a single dsRBD1, was coinjected with RNA made from a myc-tagged ADAR1 construct containing a single dsRBD2. Translation of the GFP-tagged construct was followed using appropriate antibodies. The dsRBD2-containing construct shows specific enrichment at a few chromosomal sites (D, top arrows), whereas the dsRBD1-containing construct shows a more homogeneous chromosomal staining. However, a few sites are specifically highlighted by the dsRBD1-containing constructs and, thus, appear green in the merged image (D, bottom arrows). Merged images of dsRBD1 and dsRBD2 labeling are shown (merge) as well as images of the chromosomes taken by differential interference contrast (NOM). Bar, 20 μm
Mentions: To assess the role of individual domains in chromosomal targeting, we initially compared the distribution of single domain constructs with that of the endogenous protein. In these experiments, no single domain was able to mimic endogenous staining including enrichment at the special loop. Interestingly, however, any individual domain was capable of restoring at least some level of chromosomal localization. Unlike the distribution of full-length ADAR1, constructs bearing dsRBD1 or dsRBD2 predominantly highlighted a few loops on all chromosomes (Fig. 5) , whereas the dsRBD3-containing construct showed a rather weak and relatively homogeneous staining (unpublished data). Moreover, chromosomal loops that were labeled by individual domains were always stained by the endogenous antibody, but not vice versa (Fig. 5). To ensure that the observed labeling was not due to insufficient incubation time, and thus, reduced translation product levels, oocytes were cultured for up to 10 d. Assessment at different time points showed that labeling increased slightly over the first 48-h period, but remained constant for the remainder of the experiment. Additionally, Western blots of injected oocytes were made to control for equal and sufficient protein production (unpublished data).

Bottom Line: Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally.Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA-binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein.Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell Biology and Genetics, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria.

ABSTRACT
The RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) deaminates adenosines to inosines in double-stranded RNA substrates. Currently, it is not clear how the enzyme targets and discriminates different substrates in vivo. However, it has been shown that the deaminase domain plays an important role in distinguishing various adenosines within a given substrate RNA in vitro. Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally. Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA-binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein. Most importantly, we show that individual dsRBDs are capable of recognizing different chromosomal sites in an apparently specific manner. Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

Show MeSH