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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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The dsRBDs are necessary and sufficient for chromosomal association. Lampbrush chromosome preparations were made from oocytes injected with RNA transcribed from either of two myc-tagged constructs. In vivo translation of both constructs was followed using mAb 9E10 and a secondary FITC-labeled antibody (FITC). Expression of the central part of the protein from the end of the Z-β domain up to the end of dsRBD3 (top, dsRBD1-2-3) results in normal chromosomal association and special loop enrichment. In contrast, removal of the three dsRBDs from the full-length protein (bottom, Δ dsRBDs) causes loss of chromosomal and intranuclear staining. All preparations were costained for endogenous ADAR1 using the SAT3 antiserum and detected with a secondary TRITC-labeled antibody (TRITC). Enrichment at the special loop on the third chromosome is marked by arrows. Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Bar, 20 μm.
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fig3: The dsRBDs are necessary and sufficient for chromosomal association. Lampbrush chromosome preparations were made from oocytes injected with RNA transcribed from either of two myc-tagged constructs. In vivo translation of both constructs was followed using mAb 9E10 and a secondary FITC-labeled antibody (FITC). Expression of the central part of the protein from the end of the Z-β domain up to the end of dsRBD3 (top, dsRBD1-2-3) results in normal chromosomal association and special loop enrichment. In contrast, removal of the three dsRBDs from the full-length protein (bottom, Δ dsRBDs) causes loss of chromosomal and intranuclear staining. All preparations were costained for endogenous ADAR1 using the SAT3 antiserum and detected with a secondary TRITC-labeled antibody (TRITC). Enrichment at the special loop on the third chromosome is marked by arrows. Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Bar, 20 μm.

Mentions: To test this we took two approaches. First, we deleted the three dsRBDs from the full-length myc-tagged ADAR1, fusing the ZBD, NLS, and the deaminase domain in frame. As expected, this construct failed to stain any intranuclear structures (Fig. 3) , whereas Western blots of nuclei and cytoplasms clearly showed that the construct had properly expressed and accumulated in the nucleus (unpublished data). Second, the converse experiment was performed by injecting a construct that contained only the NLS and the three dsRBDs. A fragment that spanned from the end of the ZBD, up to and including all of the third dsRBD, was myc-tagged at either end. Injection of this construct resulted in chromosomal labeling, and colocalization with the endogenous protein including enrichment at the special loop on chromosome 3 (Eckmann and Jantsch, 1999). Although this construct contained all three dsRBDs and the NLS, we were also aware that it contained the COOH terminus of the ZBD. To ensure that these extra amino acids had no effect on chromosomal targeting, the remainder of the Z-β domain was removed. Again, normal chromosomal labeling and colocalization with endogenous ADAR1 including enrichment at the special loop was observed (Fig. 3).


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)

The dsRBDs are necessary and sufficient for chromosomal association. Lampbrush chromosome preparations were made from oocytes injected with RNA transcribed from either of two myc-tagged constructs. In vivo translation of both constructs was followed using mAb 9E10 and a secondary FITC-labeled antibody (FITC). Expression of the central part of the protein from the end of the Z-β domain up to the end of dsRBD3 (top, dsRBD1-2-3) results in normal chromosomal association and special loop enrichment. In contrast, removal of the three dsRBDs from the full-length protein (bottom, Δ dsRBDs) causes loss of chromosomal and intranuclear staining. All preparations were costained for endogenous ADAR1 using the SAT3 antiserum and detected with a secondary TRITC-labeled antibody (TRITC). Enrichment at the special loop on the third chromosome is marked by arrows. Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Bar, 20 μm.
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Related In: Results  -  Collection

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fig3: The dsRBDs are necessary and sufficient for chromosomal association. Lampbrush chromosome preparations were made from oocytes injected with RNA transcribed from either of two myc-tagged constructs. In vivo translation of both constructs was followed using mAb 9E10 and a secondary FITC-labeled antibody (FITC). Expression of the central part of the protein from the end of the Z-β domain up to the end of dsRBD3 (top, dsRBD1-2-3) results in normal chromosomal association and special loop enrichment. In contrast, removal of the three dsRBDs from the full-length protein (bottom, Δ dsRBDs) causes loss of chromosomal and intranuclear staining. All preparations were costained for endogenous ADAR1 using the SAT3 antiserum and detected with a secondary TRITC-labeled antibody (TRITC). Enrichment at the special loop on the third chromosome is marked by arrows. Preparations were also stained with DAPI, and images of chromosomes were taken by differential interference contrast (NOM). Bar, 20 μm.
Mentions: To test this we took two approaches. First, we deleted the three dsRBDs from the full-length myc-tagged ADAR1, fusing the ZBD, NLS, and the deaminase domain in frame. As expected, this construct failed to stain any intranuclear structures (Fig. 3) , whereas Western blots of nuclei and cytoplasms clearly showed that the construct had properly expressed and accumulated in the nucleus (unpublished data). Second, the converse experiment was performed by injecting a construct that contained only the NLS and the three dsRBDs. A fragment that spanned from the end of the ZBD, up to and including all of the third dsRBD, was myc-tagged at either end. Injection of this construct resulted in chromosomal labeling, and colocalization with the endogenous protein including enrichment at the special loop on chromosome 3 (Eckmann and Jantsch, 1999). Although this construct contained all three dsRBDs and the NLS, we were also aware that it contained the COOH terminus of the ZBD. To ensure that these extra amino acids had no effect on chromosomal targeting, the remainder of the Z-β domain was removed. Again, normal chromosomal labeling and colocalization with endogenous ADAR1 including enrichment at the special loop was observed (Fig. 3).

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