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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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Deletion of the deaminase domain does not affect chromosomal localization. (A) RNA transcribed from full-length myc-tagged ADAR1 and a myc-tagged construct where the deaminase domain had been removed was injected individually into Xenopus oocytes. Lampbrush chromosome preparations were made and in vivo translation of each RNA was followed using the anti–myc mAb 9E10 and a secondary FITC-labeled antibody (FITC). Simultaneously, all preparations were stained for endogenous ADAR1 using the SAT3 antisera that was detected with a secondary TRITC-labeled antibody (TRITC). Injection of full-length ADAR1 (top) resulted in normal chromosomal localization compared with the endogenous protein. This was also the case when the deaminase domain was removed (bottom, Δ Deaminase), indicating at least at the chromosomal level that this domain does not play a role in targeting ADAR1 to nascent transcripts. Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Bar, 20 μm. (B) Western blot analysis of oocyte germinal vesicles (GV) and cytoplasms (CYT) from uninjected oocytes and oocytes expressing myc-tagged ADAR1 constructs to verify expression. Both myc-tagged versions express well and accumulate in the nucleus and are easily detected with the mAb 9E10. Cytoplasmic signals are seen 24 h after injection, but diminish over time as the protein is transported into the nucleus. In comparison, no signal is detected in uninjected oocytes indicating the specificity of mAb 9E10 for the injected constructs. Translated products correspond nicely to the predicted molecular masses of 175 kD (full-length ADAR1) and 130 kD (Δ Deaminase) indicated by arrows. Breakdown products can be observed at 120 kD (full-length) and 100 kD (Δ Deaminase). However, molecular mass calculations indicate that proteolytic cleavage has to occur upstream of the dsRBDs and, therefore, does not affect the RNA-binding capacity of the resulting fragments.
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fig2: Deletion of the deaminase domain does not affect chromosomal localization. (A) RNA transcribed from full-length myc-tagged ADAR1 and a myc-tagged construct where the deaminase domain had been removed was injected individually into Xenopus oocytes. Lampbrush chromosome preparations were made and in vivo translation of each RNA was followed using the anti–myc mAb 9E10 and a secondary FITC-labeled antibody (FITC). Simultaneously, all preparations were stained for endogenous ADAR1 using the SAT3 antisera that was detected with a secondary TRITC-labeled antibody (TRITC). Injection of full-length ADAR1 (top) resulted in normal chromosomal localization compared with the endogenous protein. This was also the case when the deaminase domain was removed (bottom, Δ Deaminase), indicating at least at the chromosomal level that this domain does not play a role in targeting ADAR1 to nascent transcripts. Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Bar, 20 μm. (B) Western blot analysis of oocyte germinal vesicles (GV) and cytoplasms (CYT) from uninjected oocytes and oocytes expressing myc-tagged ADAR1 constructs to verify expression. Both myc-tagged versions express well and accumulate in the nucleus and are easily detected with the mAb 9E10. Cytoplasmic signals are seen 24 h after injection, but diminish over time as the protein is transported into the nucleus. In comparison, no signal is detected in uninjected oocytes indicating the specificity of mAb 9E10 for the injected constructs. Translated products correspond nicely to the predicted molecular masses of 175 kD (full-length ADAR1) and 130 kD (Δ Deaminase) indicated by arrows. Breakdown products can be observed at 120 kD (full-length) and 100 kD (Δ Deaminase). However, molecular mass calculations indicate that proteolytic cleavage has to occur upstream of the dsRBDs and, therefore, does not affect the RNA-binding capacity of the resulting fragments.

Mentions: Removal of the deaminase domain had no effect on chromosomal localization with all chromosomes being efficiently labeled. When compared with the endogenous protein, the Δ-deaminase construct was distributed on individual chromosomes similar to endogenous ADAR1 with all loops labeled by the endogenous protein also being labeled by the injected construct. Such colocalization was also observed for the full-length construct (Fig. 2 A). Expression of injected constructs was verified by Western blotting of nuclei and cytoplasmic extracts using mAb 9E10 (Fig. 2 B). However, it should be noted that the SAT3 antiserum was generated against the NH2 terminus of Xenopus ADAR1. Therefore, the SAT3 antiserum can also detect the injected Δ-deaminase and full-length constructs. Nonetheless, SAT3 staining looked virtually identical in both injected and uninjected control oocytes (Eckmann and Jantsch, 1999; 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)

Deletion of the deaminase domain does not affect chromosomal localization. (A) RNA transcribed from full-length myc-tagged ADAR1 and a myc-tagged construct where the deaminase domain had been removed was injected individually into Xenopus oocytes. Lampbrush chromosome preparations were made and in vivo translation of each RNA was followed using the anti–myc mAb 9E10 and a secondary FITC-labeled antibody (FITC). Simultaneously, all preparations were stained for endogenous ADAR1 using the SAT3 antisera that was detected with a secondary TRITC-labeled antibody (TRITC). Injection of full-length ADAR1 (top) resulted in normal chromosomal localization compared with the endogenous protein. This was also the case when the deaminase domain was removed (bottom, Δ Deaminase), indicating at least at the chromosomal level that this domain does not play a role in targeting ADAR1 to nascent transcripts. Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Bar, 20 μm. (B) Western blot analysis of oocyte germinal vesicles (GV) and cytoplasms (CYT) from uninjected oocytes and oocytes expressing myc-tagged ADAR1 constructs to verify expression. Both myc-tagged versions express well and accumulate in the nucleus and are easily detected with the mAb 9E10. Cytoplasmic signals are seen 24 h after injection, but diminish over time as the protein is transported into the nucleus. In comparison, no signal is detected in uninjected oocytes indicating the specificity of mAb 9E10 for the injected constructs. Translated products correspond nicely to the predicted molecular masses of 175 kD (full-length ADAR1) and 130 kD (Δ Deaminase) indicated by arrows. Breakdown products can be observed at 120 kD (full-length) and 100 kD (Δ Deaminase). However, molecular mass calculations indicate that proteolytic cleavage has to occur upstream of the dsRBDs and, therefore, does not affect the RNA-binding capacity of the resulting fragments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172894&req=5

fig2: Deletion of the deaminase domain does not affect chromosomal localization. (A) RNA transcribed from full-length myc-tagged ADAR1 and a myc-tagged construct where the deaminase domain had been removed was injected individually into Xenopus oocytes. Lampbrush chromosome preparations were made and in vivo translation of each RNA was followed using the anti–myc mAb 9E10 and a secondary FITC-labeled antibody (FITC). Simultaneously, all preparations were stained for endogenous ADAR1 using the SAT3 antisera that was detected with a secondary TRITC-labeled antibody (TRITC). Injection of full-length ADAR1 (top) resulted in normal chromosomal localization compared with the endogenous protein. This was also the case when the deaminase domain was removed (bottom, Δ Deaminase), indicating at least at the chromosomal level that this domain does not play a role in targeting ADAR1 to nascent transcripts. Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Bar, 20 μm. (B) Western blot analysis of oocyte germinal vesicles (GV) and cytoplasms (CYT) from uninjected oocytes and oocytes expressing myc-tagged ADAR1 constructs to verify expression. Both myc-tagged versions express well and accumulate in the nucleus and are easily detected with the mAb 9E10. Cytoplasmic signals are seen 24 h after injection, but diminish over time as the protein is transported into the nucleus. In comparison, no signal is detected in uninjected oocytes indicating the specificity of mAb 9E10 for the injected constructs. Translated products correspond nicely to the predicted molecular masses of 175 kD (full-length ADAR1) and 130 kD (Δ Deaminase) indicated by arrows. Breakdown products can be observed at 120 kD (full-length) and 100 kD (Δ Deaminase). However, molecular mass calculations indicate that proteolytic cleavage has to occur upstream of the dsRBDs and, therefore, does not affect the RNA-binding capacity of the resulting fragments.
Mentions: Removal of the deaminase domain had no effect on chromosomal localization with all chromosomes being efficiently labeled. When compared with the endogenous protein, the Δ-deaminase construct was distributed on individual chromosomes similar to endogenous ADAR1 with all loops labeled by the endogenous protein also being labeled by the injected construct. Such colocalization was also observed for the full-length construct (Fig. 2 A). Expression of injected constructs was verified by Western blotting of nuclei and cytoplasmic extracts using mAb 9E10 (Fig. 2 B). However, it should be noted that the SAT3 antiserum was generated against the NH2 terminus of Xenopus ADAR1. Therefore, the SAT3 antiserum can also detect the injected Δ-deaminase and full-length constructs. Nonetheless, SAT3 staining looked virtually identical in both injected and uninjected control oocytes (Eckmann and Jantsch, 1999; 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
Related in: MedlinePlus