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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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In situ localization  of ADAR1.2 and ADAR1.1  deletions. (a, e, and i) DIC  image, (b, f, and j) DAPI  staining, (c, g, and k) localization of myc-tagged ADAR1  variants as seen in the fluorescein channel, and (d, h, l)  localization of endogenous  ADAR1 as seen after staining with SAT4 antiserum in  the rhodamine channel. (a–d)  myc-tagged ADAR1.2 localizes like ADAR1.1 to regular  loops and to the special loop  on bivalent no. 3. (c) the localization of myc-tagged  ADAR1.2 and (d) endogenous protein is virtually identical. (e–h) The NH2-terminal  peptide repeats found in  ADAR1.1 are not required  for localization of the protein. (g) myc-tagged ΔREP  construct localizes to nascent  transcripts and to the special  loop on bivalent no. 3. (i–l)  Construct ΔZBD, deleting  the NH2-terminal end of  ADAR1.1 including a putative Z-DNA binding domain  and one putative NLS, shows  wild-type-like in situ localization. (k) myc-tagged ΔZBD  construct (l) counterstaining with Sat4 antiserum.  Bar, 10 μm.
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Figure 6: In situ localization of ADAR1.2 and ADAR1.1 deletions. (a, e, and i) DIC image, (b, f, and j) DAPI staining, (c, g, and k) localization of myc-tagged ADAR1 variants as seen in the fluorescein channel, and (d, h, l) localization of endogenous ADAR1 as seen after staining with SAT4 antiserum in the rhodamine channel. (a–d) myc-tagged ADAR1.2 localizes like ADAR1.1 to regular loops and to the special loop on bivalent no. 3. (c) the localization of myc-tagged ADAR1.2 and (d) endogenous protein is virtually identical. (e–h) The NH2-terminal peptide repeats found in ADAR1.1 are not required for localization of the protein. (g) myc-tagged ΔREP construct localizes to nascent transcripts and to the special loop on bivalent no. 3. (i–l) Construct ΔZBD, deleting the NH2-terminal end of ADAR1.1 including a putative Z-DNA binding domain and one putative NLS, shows wild-type-like in situ localization. (k) myc-tagged ΔZBD construct (l) counterstaining with Sat4 antiserum. Bar, 10 μm.

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)

In situ localization  of ADAR1.2 and ADAR1.1  deletions. (a, e, and i) DIC  image, (b, f, and j) DAPI  staining, (c, g, and k) localization of myc-tagged ADAR1  variants as seen in the fluorescein channel, and (d, h, l)  localization of endogenous  ADAR1 as seen after staining with SAT4 antiserum in  the rhodamine channel. (a–d)  myc-tagged ADAR1.2 localizes like ADAR1.1 to regular  loops and to the special loop  on bivalent no. 3. (c) the localization of myc-tagged  ADAR1.2 and (d) endogenous protein is virtually identical. (e–h) The NH2-terminal  peptide repeats found in  ADAR1.1 are not required  for localization of the protein. (g) myc-tagged ΔREP  construct localizes to nascent  transcripts and to the special  loop on bivalent no. 3. (i–l)  Construct ΔZBD, deleting  the NH2-terminal end of  ADAR1.1 including a putative Z-DNA binding domain  and one putative NLS, shows  wild-type-like in situ localization. (k) myc-tagged ΔZBD  construct (l) counterstaining with Sat4 antiserum.  Bar, 10 μm.
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Figure 6: In situ localization of ADAR1.2 and ADAR1.1 deletions. (a, e, and i) DIC image, (b, f, and j) DAPI staining, (c, g, and k) localization of myc-tagged ADAR1 variants as seen in the fluorescein channel, and (d, h, l) localization of endogenous ADAR1 as seen after staining with SAT4 antiserum in the rhodamine channel. (a–d) myc-tagged ADAR1.2 localizes like ADAR1.1 to regular loops and to the special loop on bivalent no. 3. (c) the localization of myc-tagged ADAR1.2 and (d) endogenous protein is virtually identical. (e–h) The NH2-terminal peptide repeats found in ADAR1.1 are not required for localization of the protein. (g) myc-tagged ΔREP construct localizes to nascent transcripts and to the special loop on bivalent no. 3. (i–l) Construct ΔZBD, deleting the NH2-terminal end of ADAR1.1 including a putative Z-DNA binding domain and one putative NLS, shows wild-type-like in situ localization. (k) myc-tagged ΔZBD construct (l) counterstaining with Sat4 antiserum. Bar, 10 μm.
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.

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