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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.

Show MeSH
Schematic representation of Xenopus ADAR1 and mutant constructs used in this paper. The 1,271–amino acid Xenopus ADAR1 protein is depicted to scale at the top with mutant constructs shown underneath. Subregions of the protein are indicated as follows: REP, 11-aa peptide repeats; ZBD, Z-DNA binding domain; NLS, nuclear localization signal; dsRBDs, double-stranded RNA-binding domains; and Deaminase, catalytic deaminase domain. The ability of constructs to label chromosomes (Chr) and enrich at the special loop (Sp. loop) is shown on the right and indicated either as positive (+), negative (−), or patchy (+/−). Deletion of the deaminase domain (Δ deaminase) had no effect on chromosomal labeling and special loop enrichment. Expression of the central part of the protein from the end of the ZBD up to the end of dsRBD3 (dsRBD1-2-3) resulted in the same localization pattern as the wild-type protein, which is indicated by chromosomal association and special loop enrichment. Conversely, removal of the three dsRBDS from the full-length protein (Δ dsRBDs) resulted in a loss of all nuclear staining. Replacement of the endogenous dsRBDs with single dsRBDs (dsRBD2), duplicated dsRBDs (dsRBD2-2) or triplicated dsRBDs (dsRBD2-2-2) failed to restore normal chromosomal association. All constructs gave patchy labeling (+/−) and none enriched at the special loop (for simplicity, only dsRBD2 containing constructs are shown).
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fig1: Schematic representation of Xenopus ADAR1 and mutant constructs used in this paper. The 1,271–amino acid Xenopus ADAR1 protein is depicted to scale at the top with mutant constructs shown underneath. Subregions of the protein are indicated as follows: REP, 11-aa peptide repeats; ZBD, Z-DNA binding domain; NLS, nuclear localization signal; dsRBDs, double-stranded RNA-binding domains; and Deaminase, catalytic deaminase domain. The ability of constructs to label chromosomes (Chr) and enrich at the special loop (Sp. loop) is shown on the right and indicated either as positive (+), negative (−), or patchy (+/−). Deletion of the deaminase domain (Δ deaminase) had no effect on chromosomal labeling and special loop enrichment. Expression of the central part of the protein from the end of the ZBD up to the end of dsRBD3 (dsRBD1-2-3) resulted in the same localization pattern as the wild-type protein, which is indicated by chromosomal association and special loop enrichment. Conversely, removal of the three dsRBDS from the full-length protein (Δ dsRBDs) resulted in a loss of all nuclear staining. Replacement of the endogenous dsRBDs with single dsRBDs (dsRBD2), duplicated dsRBDs (dsRBD2-2) or triplicated dsRBDs (dsRBD2-2-2) failed to restore normal chromosomal association. All constructs gave patchy labeling (+/−) and none enriched at the special loop (for simplicity, only dsRBD2 containing constructs are shown).

Mentions: Adenosine deaminases that act on RNA (ADARs)* are a family of RNA-editing enzymes that catalyze the hydrolytic deamination of adenosines to inosines in a diverse group of mostly double-stranded RNA substrates. As inosine basepairs like guanosine, the editing event frequently leads to a codon exchange when the substrate is an mRNA (Bass, 1997). Having first been described in Xenopus oocytes and embryos, ADAR-like activity has subsequently been found in every metazoan tissue tested, and to date, three distinct ADAR enzymes are cloned and characterized from various organisms, termed ADAR1, ADAR2, and ADAR3 (Keegan et al., 2001). In addition to RNA editing, ADAR1 was recently suggested to be involved in the regulation of nuclear translation (Herbert et al., 2002). Structurally, all ADARs possess a conserved deaminase domain in their COOH terminus required for enzymatic activity as well as one or several copies of the double-stranded RNA-binding domain (dsRBD) in their central region. In addition, ADAR1 proteins have a long NH2 terminus that contains two tandemly arranged Z-DNA–binding domains (ZBDs), termed Z-α and Z-β (Keegan et al., 2001; see Fig. 1).


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)

Schematic representation of Xenopus ADAR1 and mutant constructs used in this paper. The 1,271–amino acid Xenopus ADAR1 protein is depicted to scale at the top with mutant constructs shown underneath. Subregions of the protein are indicated as follows: REP, 11-aa peptide repeats; ZBD, Z-DNA binding domain; NLS, nuclear localization signal; dsRBDs, double-stranded RNA-binding domains; and Deaminase, catalytic deaminase domain. The ability of constructs to label chromosomes (Chr) and enrich at the special loop (Sp. loop) is shown on the right and indicated either as positive (+), negative (−), or patchy (+/−). Deletion of the deaminase domain (Δ deaminase) had no effect on chromosomal labeling and special loop enrichment. Expression of the central part of the protein from the end of the ZBD up to the end of dsRBD3 (dsRBD1-2-3) resulted in the same localization pattern as the wild-type protein, which is indicated by chromosomal association and special loop enrichment. Conversely, removal of the three dsRBDS from the full-length protein (Δ dsRBDs) resulted in a loss of all nuclear staining. Replacement of the endogenous dsRBDs with single dsRBDs (dsRBD2), duplicated dsRBDs (dsRBD2-2) or triplicated dsRBDs (dsRBD2-2-2) failed to restore normal chromosomal association. All constructs gave patchy labeling (+/−) and none enriched at the special loop (for simplicity, only dsRBD2 containing constructs are shown).
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Related In: Results  -  Collection

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fig1: Schematic representation of Xenopus ADAR1 and mutant constructs used in this paper. The 1,271–amino acid Xenopus ADAR1 protein is depicted to scale at the top with mutant constructs shown underneath. Subregions of the protein are indicated as follows: REP, 11-aa peptide repeats; ZBD, Z-DNA binding domain; NLS, nuclear localization signal; dsRBDs, double-stranded RNA-binding domains; and Deaminase, catalytic deaminase domain. The ability of constructs to label chromosomes (Chr) and enrich at the special loop (Sp. loop) is shown on the right and indicated either as positive (+), negative (−), or patchy (+/−). Deletion of the deaminase domain (Δ deaminase) had no effect on chromosomal labeling and special loop enrichment. Expression of the central part of the protein from the end of the ZBD up to the end of dsRBD3 (dsRBD1-2-3) resulted in the same localization pattern as the wild-type protein, which is indicated by chromosomal association and special loop enrichment. Conversely, removal of the three dsRBDS from the full-length protein (Δ dsRBDs) resulted in a loss of all nuclear staining. Replacement of the endogenous dsRBDs with single dsRBDs (dsRBD2), duplicated dsRBDs (dsRBD2-2) or triplicated dsRBDs (dsRBD2-2-2) failed to restore normal chromosomal association. All constructs gave patchy labeling (+/−) and none enriched at the special loop (for simplicity, only dsRBD2 containing constructs are shown).
Mentions: Adenosine deaminases that act on RNA (ADARs)* are a family of RNA-editing enzymes that catalyze the hydrolytic deamination of adenosines to inosines in a diverse group of mostly double-stranded RNA substrates. As inosine basepairs like guanosine, the editing event frequently leads to a codon exchange when the substrate is an mRNA (Bass, 1997). Having first been described in Xenopus oocytes and embryos, ADAR-like activity has subsequently been found in every metazoan tissue tested, and to date, three distinct ADAR enzymes are cloned and characterized from various organisms, termed ADAR1, ADAR2, and ADAR3 (Keegan et al., 2001). In addition to RNA editing, ADAR1 was recently suggested to be involved in the regulation of nuclear translation (Herbert et al., 2002). Structurally, all ADARs possess a conserved deaminase domain in their COOH terminus required for enzymatic activity as well as one or several copies of the double-stranded RNA-binding domain (dsRBD) in their central region. In addition, ADAR1 proteins have a long NH2 terminus that contains two tandemly arranged Z-DNA–binding domains (ZBDs), termed Z-α and Z-β (Keegan et al., 2001; see Fig. 1).

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