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The RNA-editing enzyme ADAR1 is localized to the nascent ribonucleoprotein matrix on Xenopus lampbrush chromosomes but specifically associates with an atypical loop.

Eckmann CR, Jantsch MF - J. Cell Biol. (1999)

Bottom Line: We demonstrate that both variants of the enzyme are associated with transcriptionally active chromosome loops suggesting that the enzyme acts cotranscriptionally.Inhibition of splicing, another cotranscriptional process, does not affect the chromosomal localization of ADAR1.Finally, mutational analysis of ADAR1 demonstrates that a putative Z-DNA binding domain present in ADAR1 is not required for chromosomal targeting of the protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Cytology and Genetics, Institute of Botany, University of Vienna, A-1030 Vienna, Austria.

ABSTRACT
Double-stranded RNA adenosine deaminase (ADAR1, dsRAD, DRADA) converts adenosines to inosines in double-stranded RNAs. Few candidate substrates for ADAR1 editing are known at this point and it is not known how substrate recognition is achieved. In some cases editing sites are defined by basepaired regions formed between intronic and exonic sequences, suggesting that the enzyme might function cotranscriptionally. We have isolated two variants of Xenopus laevis ADAR1 for which no editing substrates are currently known. We demonstrate that both variants of the enzyme are associated with transcriptionally active chromosome loops suggesting that the enzyme acts cotranscriptionally. The widespread distribution of the protein along the entire chromosome indicates that ADAR1 associates with the RNP matrix in a substrate-independent manner. Inhibition of splicing, another cotranscriptional process, does not affect the chromosomal localization of ADAR1. Furthermore, we can show that the enzyme is dramatically enriched on a special RNA-containing loop that seems transcriptionally silent. Detailed analysis of this loop suggests that it might represent a site of ADAR1 storage or a site where active RNA editing is taking place. Finally, mutational analysis of ADAR1 demonstrates that a putative Z-DNA binding domain present in ADAR1 is not required for chromosomal targeting of the protein.

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Xenopus ADAR1.1 and ADAR1.2 are recognized by  SAT antisera. (a) Schematic representation of myc-tagged Xenopus ADAR1.1 and ADAR1.2 proteins. ADAR1.1 contains an  11–amino acid long peptide motif that is repeated 14 times (left  striped box). Both proteins contain a putative Z-DNA binding  domain (black box), three dsRBDs (right striped boxes) and a  catalytic deamination domain (light gray box). The positions of  two putative NLSs is indicated by asterisks. ADAR1.1 was myc-tagged at the NH2 terminus, the COOH terminus or at both ends  while ADAR1.2 was only myc-tagged at its COOH terminus. An  AUG codon was introduced at the 5′ end of the ADAR1.2  cDNA that was missing from our original cDNA clone. (b) Western blots of oocyte nuclei (GV), cytoplasms (C), and XlA6 cells  (TC) detected with Sat3 or Sat4 antisera directed against part of  the ADAR1.1 protein. Both antisera recognize a nuclear protein  of 125 kD that can also be detected in tissue culture cells. The detected band is smaller than the predicted molecular mass of  ADAR1.1 but correlates well with the reported molecular mass  of purified ADAR1.1 from Xenopus. 5 GVs and 5 cytoplasms  were loaded per lane. (c) Sat antisera can precipitate ADAR1.1  and ADAR1.2. Myc-tagged ADAR1.1 or ADAR1.2 was expressed in Xenopus oocytes. Oocyte extracts expressing either  ADAR protein were used for immunoprecipitation with Sat3 antiserum (IS), preimmune serum (PI), or Sat4 antiserum (not  shown). The precipitated material was probed with mAb 9E10  for the presence of myc-tagged ADAR1. Both ADAR1.1 and  ADAR1.2 could be precipitated by Sat antiserum (IS) but not  by the corresponding preimmune serum (PI). Positions of myc-tagged ADAR1.1 and ADAR1.2 are indicated by arrowheads.  myc-tagged ADAR1.1 can be detected in its full-length form  (185 kD) and in a smaller version (150 kD) which is probably a  proteolytic breakdown product.
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Figure 1: Xenopus ADAR1.1 and ADAR1.2 are recognized by SAT antisera. (a) Schematic representation of myc-tagged Xenopus ADAR1.1 and ADAR1.2 proteins. ADAR1.1 contains an 11–amino acid long peptide motif that is repeated 14 times (left striped box). Both proteins contain a putative Z-DNA binding domain (black box), three dsRBDs (right striped boxes) and a catalytic deamination domain (light gray box). The positions of two putative NLSs is indicated by asterisks. ADAR1.1 was myc-tagged at the NH2 terminus, the COOH terminus or at both ends while ADAR1.2 was only myc-tagged at its COOH terminus. An AUG codon was introduced at the 5′ end of the ADAR1.2 cDNA that was missing from our original cDNA clone. (b) Western blots of oocyte nuclei (GV), cytoplasms (C), and XlA6 cells (TC) detected with Sat3 or Sat4 antisera directed against part of the ADAR1.1 protein. Both antisera recognize a nuclear protein of 125 kD that can also be detected in tissue culture cells. The detected band is smaller than the predicted molecular mass of ADAR1.1 but correlates well with the reported molecular mass of purified ADAR1.1 from Xenopus. 5 GVs and 5 cytoplasms were loaded per lane. (c) Sat antisera can precipitate ADAR1.1 and ADAR1.2. Myc-tagged ADAR1.1 or ADAR1.2 was expressed in Xenopus oocytes. Oocyte extracts expressing either ADAR protein were used for immunoprecipitation with Sat3 antiserum (IS), preimmune serum (PI), or Sat4 antiserum (not shown). The precipitated material was probed with mAb 9E10 for the presence of myc-tagged ADAR1. Both ADAR1.1 and ADAR1.2 could be precipitated by Sat antiserum (IS) but not by the corresponding preimmune serum (PI). Positions of myc-tagged ADAR1.1 and ADAR1.2 are indicated by arrowheads. myc-tagged ADAR1.1 can be detected in its full-length form (185 kD) and in a smaller version (150 kD) which is probably a proteolytic breakdown product.

Mentions: We have recently cloned two closely related variants of Xenopus laevis ADAR1 from an ovary cDNA library (Brooks et al., 1998). The two cDNAs are, with the exception of a few exchanges, virtually identical to the two cDNAs isolated by Bass and coworkers which were first termed dsRAD-1 and dsRAD-2 but have since been renamed ADAR1.1 and ADAR1.2, respectively (Hough and Bass, 1997; Bass et al., 1997). Both proteins show a high degree of sequence identity in their central and COOH-terminal regions where they contain three dsRBDs and a conserved deamination domain, respectively. However, at the NH2 terminus the two proteins show marked differences. ADAR1.1 contains 14 repeats of an 11–amino acids long sequence motif that is only present in one copy in ADAR1.2 (see Fig. 1 a). ADAR1.1 is a protein of 1,270 amino acids whereas no proper AUG start codon has been determined for ADAR1.2. However, Northern blots indicate that both proteins are well expressed and seem to encode proteins of similar molecular mass (Hough and Bass, 1997).


The RNA-editing enzyme ADAR1 is localized to the nascent ribonucleoprotein matrix on Xenopus lampbrush chromosomes but specifically associates with an atypical loop.

Eckmann CR, Jantsch MF - J. Cell Biol. (1999)

Xenopus ADAR1.1 and ADAR1.2 are recognized by  SAT antisera. (a) Schematic representation of myc-tagged Xenopus ADAR1.1 and ADAR1.2 proteins. ADAR1.1 contains an  11–amino acid long peptide motif that is repeated 14 times (left  striped box). Both proteins contain a putative Z-DNA binding  domain (black box), three dsRBDs (right striped boxes) and a  catalytic deamination domain (light gray box). The positions of  two putative NLSs is indicated by asterisks. ADAR1.1 was myc-tagged at the NH2 terminus, the COOH terminus or at both ends  while ADAR1.2 was only myc-tagged at its COOH terminus. An  AUG codon was introduced at the 5′ end of the ADAR1.2  cDNA that was missing from our original cDNA clone. (b) Western blots of oocyte nuclei (GV), cytoplasms (C), and XlA6 cells  (TC) detected with Sat3 or Sat4 antisera directed against part of  the ADAR1.1 protein. Both antisera recognize a nuclear protein  of 125 kD that can also be detected in tissue culture cells. The detected band is smaller than the predicted molecular mass of  ADAR1.1 but correlates well with the reported molecular mass  of purified ADAR1.1 from Xenopus. 5 GVs and 5 cytoplasms  were loaded per lane. (c) Sat antisera can precipitate ADAR1.1  and ADAR1.2. Myc-tagged ADAR1.1 or ADAR1.2 was expressed in Xenopus oocytes. Oocyte extracts expressing either  ADAR protein were used for immunoprecipitation with Sat3 antiserum (IS), preimmune serum (PI), or Sat4 antiserum (not  shown). The precipitated material was probed with mAb 9E10  for the presence of myc-tagged ADAR1. Both ADAR1.1 and  ADAR1.2 could be precipitated by Sat antiserum (IS) but not  by the corresponding preimmune serum (PI). Positions of myc-tagged ADAR1.1 and ADAR1.2 are indicated by arrowheads.  myc-tagged ADAR1.1 can be detected in its full-length form  (185 kD) and in a smaller version (150 kD) which is probably a  proteolytic breakdown product.
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Related In: Results  -  Collection

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Figure 1: Xenopus ADAR1.1 and ADAR1.2 are recognized by SAT antisera. (a) Schematic representation of myc-tagged Xenopus ADAR1.1 and ADAR1.2 proteins. ADAR1.1 contains an 11–amino acid long peptide motif that is repeated 14 times (left striped box). Both proteins contain a putative Z-DNA binding domain (black box), three dsRBDs (right striped boxes) and a catalytic deamination domain (light gray box). The positions of two putative NLSs is indicated by asterisks. ADAR1.1 was myc-tagged at the NH2 terminus, the COOH terminus or at both ends while ADAR1.2 was only myc-tagged at its COOH terminus. An AUG codon was introduced at the 5′ end of the ADAR1.2 cDNA that was missing from our original cDNA clone. (b) Western blots of oocyte nuclei (GV), cytoplasms (C), and XlA6 cells (TC) detected with Sat3 or Sat4 antisera directed against part of the ADAR1.1 protein. Both antisera recognize a nuclear protein of 125 kD that can also be detected in tissue culture cells. The detected band is smaller than the predicted molecular mass of ADAR1.1 but correlates well with the reported molecular mass of purified ADAR1.1 from Xenopus. 5 GVs and 5 cytoplasms were loaded per lane. (c) Sat antisera can precipitate ADAR1.1 and ADAR1.2. Myc-tagged ADAR1.1 or ADAR1.2 was expressed in Xenopus oocytes. Oocyte extracts expressing either ADAR protein were used for immunoprecipitation with Sat3 antiserum (IS), preimmune serum (PI), or Sat4 antiserum (not shown). The precipitated material was probed with mAb 9E10 for the presence of myc-tagged ADAR1. Both ADAR1.1 and ADAR1.2 could be precipitated by Sat antiserum (IS) but not by the corresponding preimmune serum (PI). Positions of myc-tagged ADAR1.1 and ADAR1.2 are indicated by arrowheads. myc-tagged ADAR1.1 can be detected in its full-length form (185 kD) and in a smaller version (150 kD) which is probably a proteolytic breakdown product.
Mentions: We have recently cloned two closely related variants of Xenopus laevis ADAR1 from an ovary cDNA library (Brooks et al., 1998). The two cDNAs are, with the exception of a few exchanges, virtually identical to the two cDNAs isolated by Bass and coworkers which were first termed dsRAD-1 and dsRAD-2 but have since been renamed ADAR1.1 and ADAR1.2, respectively (Hough and Bass, 1997; Bass et al., 1997). Both proteins show a high degree of sequence identity in their central and COOH-terminal regions where they contain three dsRBDs and a conserved deamination domain, respectively. However, at the NH2 terminus the two proteins show marked differences. ADAR1.1 contains 14 repeats of an 11–amino acids long sequence motif that is only present in one copy in ADAR1.2 (see Fig. 1 a). ADAR1.1 is a protein of 1,270 amino acids whereas no proper AUG start codon has been determined for ADAR1.2. However, Northern blots indicate that both proteins are well expressed and seem to encode proteins of similar molecular mass (Hough and Bass, 1997).

Bottom Line: We demonstrate that both variants of the enzyme are associated with transcriptionally active chromosome loops suggesting that the enzyme acts cotranscriptionally.Inhibition of splicing, another cotranscriptional process, does not affect the chromosomal localization of ADAR1.Finally, mutational analysis of ADAR1 demonstrates that a putative Z-DNA binding domain present in ADAR1 is not required for chromosomal targeting of the protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Cytology and Genetics, Institute of Botany, University of Vienna, A-1030 Vienna, Austria.

ABSTRACT
Double-stranded RNA adenosine deaminase (ADAR1, dsRAD, DRADA) converts adenosines to inosines in double-stranded RNAs. Few candidate substrates for ADAR1 editing are known at this point and it is not known how substrate recognition is achieved. In some cases editing sites are defined by basepaired regions formed between intronic and exonic sequences, suggesting that the enzyme might function cotranscriptionally. We have isolated two variants of Xenopus laevis ADAR1 for which no editing substrates are currently known. We demonstrate that both variants of the enzyme are associated with transcriptionally active chromosome loops suggesting that the enzyme acts cotranscriptionally. The widespread distribution of the protein along the entire chromosome indicates that ADAR1 associates with the RNP matrix in a substrate-independent manner. Inhibition of splicing, another cotranscriptional process, does not affect the chromosomal localization of ADAR1. Furthermore, we can show that the enzyme is dramatically enriched on a special RNA-containing loop that seems transcriptionally silent. Detailed analysis of this loop suggests that it might represent a site of ADAR1 storage or a site where active RNA editing is taking place. Finally, mutational analysis of ADAR1 demonstrates that a putative Z-DNA binding domain present in ADAR1 is not required for chromosomal targeting of the protein.

Show MeSH