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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 target ADAR1 to different subsets of loops. Double injections of RNA from different myc- and GFP triplication constructs are shown as indicated. Translation of the myc constructs was detected using mAb 9E10 and a secondary TRITC-labeled antibody (TRITC), whereas the GFP constructs were detected using an anti–rabbit GFP antibody and a secondary FITC-labeled antibody (FITC). Merged enlargements are shown between (merge). (A) Injection of identical constructs label the same chromosomal sites. Shown is a double injection of RNA made from dsRBD1 triplications that were either myc- or GFP-tagged. Both tagged versions label the chromosomes equally well and target to the same sites and loops (e.g., arrows). Such a complete overlap becomes even more apparent when similar regions of the chromosome from each channel (boxed) are enlarged, colored, and merged. (B and C) Different dsRBDs target ADAR1 to different chromosomal sites. RNA made from GFP-tagged dsRBD1 triplication was injected into oocytes followed by injection of RNA either from myc-tagged dsRBD2 triplication (B) or myc-tagged dsRBD3 triplication (C). Two different representative chromosomal sites are shown in B. Each construct labels chromosomes differently with some sites being labeled by one construct not being labeled by the other (arrows). Consequently, in the merged images, there is no complete overlap of staining. Together, this demonstrates the capability of individual dsRBDs specifically targeting different transcription units. Images of chromosomes taken by differential interference contrast (NOM) are also shown. Bar, 10 μm. (D) Western blot analysis of oocyte germinal vesicles (GV) and cytoplasms (CYT) from uninjected oocytes and oocytes expressing myc- and GFP-tagged ADAR1 constructs shown in A–C to verify expression. Translated products correspond nicely to the predicted molecular masses of ∼180 kD (arrows).
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fig6: Individual dsRBDs can target ADAR1 to different subsets of loops. Double injections of RNA from different myc- and GFP triplication constructs are shown as indicated. Translation of the myc constructs was detected using mAb 9E10 and a secondary TRITC-labeled antibody (TRITC), whereas the GFP constructs were detected using an anti–rabbit GFP antibody and a secondary FITC-labeled antibody (FITC). Merged enlargements are shown between (merge). (A) Injection of identical constructs label the same chromosomal sites. Shown is a double injection of RNA made from dsRBD1 triplications that were either myc- or GFP-tagged. Both tagged versions label the chromosomes equally well and target to the same sites and loops (e.g., arrows). Such a complete overlap becomes even more apparent when similar regions of the chromosome from each channel (boxed) are enlarged, colored, and merged. (B and C) Different dsRBDs target ADAR1 to different chromosomal sites. RNA made from GFP-tagged dsRBD1 triplication was injected into oocytes followed by injection of RNA either from myc-tagged dsRBD2 triplication (B) or myc-tagged dsRBD3 triplication (C). Two different representative chromosomal sites are shown in B. Each construct labels chromosomes differently with some sites being labeled by one construct not being labeled by the other (arrows). Consequently, in the merged images, there is no complete overlap of staining. Together, this demonstrates the capability of individual dsRBDs specifically targeting different transcription units. Images of chromosomes taken by differential interference contrast (NOM) are also shown. Bar, 10 μm. (D) Western blot analysis of oocyte germinal vesicles (GV) and cytoplasms (CYT) from uninjected oocytes and oocytes expressing myc- and GFP-tagged ADAR1 constructs shown in A–C to verify expression. Translated products correspond nicely to the predicted molecular masses of ∼180 kD (arrows).

Mentions: Thus far, our experiments could demonstrate that individual dsRBDs are capable of targeting specific chromosomal loci. Moreover, increasing the level of RNA binding slightly increased chromosomal association of the protein, but it remained restricted to a few sites and failed to restore homogeneous chromosomal localization comparable to that of wild-type ADAR1. Next, we wanted to determine whether individual dsRBDs can target to different transcripts in vivo. To accomplish this, constructs containing different dsRBDs were tagged with either GFP or myc, injected, and subsequently detected within the same oocyte. To verify that the different tags did not alter the localization of the resulting fusion proteins, we initially coinjected identical myc- and GFP-tagged constructs and compared their localization to each other. Additionally, synthesis and nuclear accumulation of the resulting constructs was monitored by Western blotting of hand-enucleated oocytes and isolated germinal vesicles. As expected, the different tagging systems showed no adverse effect on the chromosomal distribution of the resulting fusion proteins, leading to a complete overlap in the localization of both myc- and GFP-tagged protein (Fig. 6) .


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 target ADAR1 to different subsets of loops. Double injections of RNA from different myc- and GFP triplication constructs are shown as indicated. Translation of the myc constructs was detected using mAb 9E10 and a secondary TRITC-labeled antibody (TRITC), whereas the GFP constructs were detected using an anti–rabbit GFP antibody and a secondary FITC-labeled antibody (FITC). Merged enlargements are shown between (merge). (A) Injection of identical constructs label the same chromosomal sites. Shown is a double injection of RNA made from dsRBD1 triplications that were either myc- or GFP-tagged. Both tagged versions label the chromosomes equally well and target to the same sites and loops (e.g., arrows). Such a complete overlap becomes even more apparent when similar regions of the chromosome from each channel (boxed) are enlarged, colored, and merged. (B and C) Different dsRBDs target ADAR1 to different chromosomal sites. RNA made from GFP-tagged dsRBD1 triplication was injected into oocytes followed by injection of RNA either from myc-tagged dsRBD2 triplication (B) or myc-tagged dsRBD3 triplication (C). Two different representative chromosomal sites are shown in B. Each construct labels chromosomes differently with some sites being labeled by one construct not being labeled by the other (arrows). Consequently, in the merged images, there is no complete overlap of staining. Together, this demonstrates the capability of individual dsRBDs specifically targeting different transcription units. Images of chromosomes taken by differential interference contrast (NOM) are also shown. Bar, 10 μm. (D) Western blot analysis of oocyte germinal vesicles (GV) and cytoplasms (CYT) from uninjected oocytes and oocytes expressing myc- and GFP-tagged ADAR1 constructs shown in A–C to verify expression. Translated products correspond nicely to the predicted molecular masses of ∼180 kD (arrows).
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Related In: Results  -  Collection

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fig6: Individual dsRBDs can target ADAR1 to different subsets of loops. Double injections of RNA from different myc- and GFP triplication constructs are shown as indicated. Translation of the myc constructs was detected using mAb 9E10 and a secondary TRITC-labeled antibody (TRITC), whereas the GFP constructs were detected using an anti–rabbit GFP antibody and a secondary FITC-labeled antibody (FITC). Merged enlargements are shown between (merge). (A) Injection of identical constructs label the same chromosomal sites. Shown is a double injection of RNA made from dsRBD1 triplications that were either myc- or GFP-tagged. Both tagged versions label the chromosomes equally well and target to the same sites and loops (e.g., arrows). Such a complete overlap becomes even more apparent when similar regions of the chromosome from each channel (boxed) are enlarged, colored, and merged. (B and C) Different dsRBDs target ADAR1 to different chromosomal sites. RNA made from GFP-tagged dsRBD1 triplication was injected into oocytes followed by injection of RNA either from myc-tagged dsRBD2 triplication (B) or myc-tagged dsRBD3 triplication (C). Two different representative chromosomal sites are shown in B. Each construct labels chromosomes differently with some sites being labeled by one construct not being labeled by the other (arrows). Consequently, in the merged images, there is no complete overlap of staining. Together, this demonstrates the capability of individual dsRBDs specifically targeting different transcription units. Images of chromosomes taken by differential interference contrast (NOM) are also shown. Bar, 10 μm. (D) Western blot analysis of oocyte germinal vesicles (GV) and cytoplasms (CYT) from uninjected oocytes and oocytes expressing myc- and GFP-tagged ADAR1 constructs shown in A–C to verify expression. Translated products correspond nicely to the predicted molecular masses of ∼180 kD (arrows).
Mentions: Thus far, our experiments could demonstrate that individual dsRBDs are capable of targeting specific chromosomal loci. Moreover, increasing the level of RNA binding slightly increased chromosomal association of the protein, but it remained restricted to a few sites and failed to restore homogeneous chromosomal localization comparable to that of wild-type ADAR1. Next, we wanted to determine whether individual dsRBDs can target to different transcripts in vivo. To accomplish this, constructs containing different dsRBDs were tagged with either GFP or myc, injected, and subsequently detected within the same oocyte. To verify that the different tags did not alter the localization of the resulting fusion proteins, we initially coinjected identical myc- and GFP-tagged constructs and compared their localization to each other. Additionally, synthesis and nuclear accumulation of the resulting constructs was monitored by Western blotting of hand-enucleated oocytes and isolated germinal vesicles. As expected, the different tagging systems showed no adverse effect on the chromosomal distribution of the resulting fusion proteins, leading to a complete overlap in the localization of both myc- and GFP-tagged protein (Fig. 6) .

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
Related in: MedlinePlus