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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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(a) Schematic representation of deletion constructs  used in this study. The peptide repeats (left striped box), the  ZBD (black box), the dsRBDs (right striped box) and the deamination domain (gray box) are indicated. ΔREP deletes the NH2-terminal peptide repeats from ADAR1.1 leaving a single,  COOH-terminal myc tag. ΔZBD deletes a longer portion from  the NH2 terminus of ADAR1.1 including the putative Z-DNA  binding domain and one of two putative NLSs. ADAR1.2 does  not contain the peptide repeats and is only myc-tagged at its  COOH terminus. An artificial AUG codon was introduced at the  beginning of the cDNA. (b) Western blot of oocyte nuclei (GV)  and cytoplasms (Cytopl.) of uninjected oocytes (−) and of oocytes expressing myc-tagged ADAR1 versions. All myc-tagged  ADAR1 versions express well and accumulate in the nucleus.  Cytoplasmic signals are seen 24 h after injection but diminish  over time as the protein is transported to the nucleus. (c)  ADAR1 undergoes NH2-terminal proteolytic cleavage. Western  blot of oocyte nuclei (GV) and cytoplasms (Cytopl.) of uninjected (−) or injected oocytes expressing ADAR1.1 myc tagged  at its COOH terminus (C), COOH and NH2 terminus (NC) or  NH2 terminus only (N). In the cytoplasm full-length versions (180  to 190 kD) of all three constructs can be easily detected (upper  two arrowheads). A smaller (150 kD) breakdown product can  only be detected for the two versions carrying a COOH-terminal  myc-tag (lower arrowhead). In the nucleus (GV) the breakdown  product is the most prominent form found 24 h after injection.  However, also the full-length versions can be detected in the nucleus. If the myc-tag is only present at the NH2 terminus (N) only  full-length protein can be detected, indicating that the NH2 terminus undergoes proteolytic cleavage.
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Figure 7: (a) Schematic representation of deletion constructs used in this study. The peptide repeats (left striped box), the ZBD (black box), the dsRBDs (right striped box) and the deamination domain (gray box) are indicated. ΔREP deletes the NH2-terminal peptide repeats from ADAR1.1 leaving a single, COOH-terminal myc tag. ΔZBD deletes a longer portion from the NH2 terminus of ADAR1.1 including the putative Z-DNA binding domain and one of two putative NLSs. ADAR1.2 does not contain the peptide repeats and is only myc-tagged at its COOH terminus. An artificial AUG codon was introduced at the beginning of the cDNA. (b) Western blot of oocyte nuclei (GV) and cytoplasms (Cytopl.) of uninjected oocytes (−) and of oocytes expressing myc-tagged ADAR1 versions. All myc-tagged ADAR1 versions express well and accumulate in the nucleus. Cytoplasmic signals are seen 24 h after injection but diminish over time as the protein is transported to the nucleus. (c) ADAR1 undergoes NH2-terminal proteolytic cleavage. Western blot of oocyte nuclei (GV) and cytoplasms (Cytopl.) of uninjected (−) or injected oocytes expressing ADAR1.1 myc tagged at its COOH terminus (C), COOH and NH2 terminus (NC) or NH2 terminus only (N). In the cytoplasm full-length versions (180 to 190 kD) of all three constructs can be easily detected (upper two arrowheads). A smaller (150 kD) breakdown product can only be detected for the two versions carrying a COOH-terminal myc-tag (lower arrowhead). In the nucleus (GV) the breakdown product is the most prominent form found 24 h after injection. However, also the full-length versions can be detected in the nucleus. If the myc-tag is only present at the NH2 terminus (N) only full-length protein can be detected, indicating that the NH2 terminus undergoes proteolytic cleavage.

Mentions: Although ADAR1.1 and ADAR1.2 show a high degree of sequence identity in their central region and at their COOH termini, their NH2 termini differ considerably (Hough and Bass, 1997). Part of this difference can be attributed to the presence of an 11–amino acids long repeat that is present in 14 almost perfect tandemly arranged copies in ADAR1.1 but only in a single copy in ADAR1.2. To test whether these peptide repeats are required for proper association of ADAR1 with the RNP matrix we have analyzed the nuclear distribution of myc-tagged ADAR1.2 and ADAR1.1 from which the NH2-terminal end including the peptide repeats had been removed (construct ΔREP, Fig. 7). When compared for their in situ localization on Xenopus LBCs, both clones showed an identical distribution: Strong labeling of the special loop was observed whereas moderate labeling was detectable on all other loops (Fig. 6). This indicates that the peptide repeats have no influence on the intranuclear association of ADAR1 with chromosome loops.


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)

(a) Schematic representation of deletion constructs  used in this study. The peptide repeats (left striped box), the  ZBD (black box), the dsRBDs (right striped box) and the deamination domain (gray box) are indicated. ΔREP deletes the NH2-terminal peptide repeats from ADAR1.1 leaving a single,  COOH-terminal myc tag. ΔZBD deletes a longer portion from  the NH2 terminus of ADAR1.1 including the putative Z-DNA  binding domain and one of two putative NLSs. ADAR1.2 does  not contain the peptide repeats and is only myc-tagged at its  COOH terminus. An artificial AUG codon was introduced at the  beginning of the cDNA. (b) Western blot of oocyte nuclei (GV)  and cytoplasms (Cytopl.) of uninjected oocytes (−) and of oocytes expressing myc-tagged ADAR1 versions. All myc-tagged  ADAR1 versions express well and accumulate in the nucleus.  Cytoplasmic signals are seen 24 h after injection but diminish  over time as the protein is transported to the nucleus. (c)  ADAR1 undergoes NH2-terminal proteolytic cleavage. Western  blot of oocyte nuclei (GV) and cytoplasms (Cytopl.) of uninjected (−) or injected oocytes expressing ADAR1.1 myc tagged  at its COOH terminus (C), COOH and NH2 terminus (NC) or  NH2 terminus only (N). In the cytoplasm full-length versions (180  to 190 kD) of all three constructs can be easily detected (upper  two arrowheads). A smaller (150 kD) breakdown product can  only be detected for the two versions carrying a COOH-terminal  myc-tag (lower arrowhead). In the nucleus (GV) the breakdown  product is the most prominent form found 24 h after injection.  However, also the full-length versions can be detected in the nucleus. If the myc-tag is only present at the NH2 terminus (N) only  full-length protein can be detected, indicating that the NH2 terminus undergoes proteolytic cleavage.
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Related In: Results  -  Collection

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Figure 7: (a) Schematic representation of deletion constructs used in this study. The peptide repeats (left striped box), the ZBD (black box), the dsRBDs (right striped box) and the deamination domain (gray box) are indicated. ΔREP deletes the NH2-terminal peptide repeats from ADAR1.1 leaving a single, COOH-terminal myc tag. ΔZBD deletes a longer portion from the NH2 terminus of ADAR1.1 including the putative Z-DNA binding domain and one of two putative NLSs. ADAR1.2 does not contain the peptide repeats and is only myc-tagged at its COOH terminus. An artificial AUG codon was introduced at the beginning of the cDNA. (b) Western blot of oocyte nuclei (GV) and cytoplasms (Cytopl.) of uninjected oocytes (−) and of oocytes expressing myc-tagged ADAR1 versions. All myc-tagged ADAR1 versions express well and accumulate in the nucleus. Cytoplasmic signals are seen 24 h after injection but diminish over time as the protein is transported to the nucleus. (c) ADAR1 undergoes NH2-terminal proteolytic cleavage. Western blot of oocyte nuclei (GV) and cytoplasms (Cytopl.) of uninjected (−) or injected oocytes expressing ADAR1.1 myc tagged at its COOH terminus (C), COOH and NH2 terminus (NC) or NH2 terminus only (N). In the cytoplasm full-length versions (180 to 190 kD) of all three constructs can be easily detected (upper two arrowheads). A smaller (150 kD) breakdown product can only be detected for the two versions carrying a COOH-terminal myc-tag (lower arrowhead). In the nucleus (GV) the breakdown product is the most prominent form found 24 h after injection. However, also the full-length versions can be detected in the nucleus. If the myc-tag is only present at the NH2 terminus (N) only full-length protein can be detected, indicating that the NH2 terminus undergoes proteolytic cleavage.
Mentions: Although ADAR1.1 and ADAR1.2 show a high degree of sequence identity in their central region and at their COOH termini, their NH2 termini differ considerably (Hough and Bass, 1997). Part of this difference can be attributed to the presence of an 11–amino acids long repeat that is present in 14 almost perfect tandemly arranged copies in ADAR1.1 but only in a single copy in ADAR1.2. To test whether these peptide repeats are required for proper association of ADAR1 with the RNP matrix we have analyzed the nuclear distribution of myc-tagged ADAR1.2 and ADAR1.1 from which the NH2-terminal end including the peptide repeats had been removed (construct ΔREP, Fig. 7). When compared for their in situ localization on Xenopus LBCs, both clones showed an identical distribution: Strong labeling of the special loop was observed whereas moderate labeling was detectable on all other loops (Fig. 6). This indicates that the peptide repeats have no influence on the intranuclear association of ADAR1 with chromosome loops.

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