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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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xlADAR1 localizes to the nascent RNP matrix and is specifically enriched on a special loop on  bivalent no. 3. LBCs were  prepared from oocytes  expressing myc-tagged  ADAR1.1. (a–d) Normal  LBC and (e–f) bivalent no. 3.  (a and e) DIC image, (b and  f) DAPI staining, (c and g)  localization of endogenous  ADAR1 detected with SAT4  antiserum in the rhodamine  channel and (d and h) localization of myc-tagged  ADAR1.1 detected with  mAb 9E10 in the fluorescein  channel. Endogenous and ectopically expressed ADAR1  is localized to LBC loops. (g  and h) A special loop on  bivalent no. 3 is enriched for  endogenous (g) and ectopically expressed ADAR1 (h).  The special loop is marked  by arrows. Faint background  signals can be seen on nucleoli (N) or snurposomes (S)  by staining with SAT4 antiserum that is not seen with  myc-tagged ADAR1.1. Bar,  10 μm.
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Figure 2: xlADAR1 localizes to the nascent RNP matrix and is specifically enriched on a special loop on bivalent no. 3. LBCs were prepared from oocytes expressing myc-tagged ADAR1.1. (a–d) Normal LBC and (e–f) bivalent no. 3. (a and e) DIC image, (b and f) DAPI staining, (c and g) localization of endogenous ADAR1 detected with SAT4 antiserum in the rhodamine channel and (d and h) localization of myc-tagged ADAR1.1 detected with mAb 9E10 in the fluorescein channel. Endogenous and ectopically expressed ADAR1 is localized to LBC loops. (g and h) A special loop on bivalent no. 3 is enriched for endogenous (g) and ectopically expressed ADAR1 (h). The special loop is marked by arrows. Faint background signals can be seen on nucleoli (N) or snurposomes (S) by staining with SAT4 antiserum that is not seen with myc-tagged ADAR1.1. Bar, 10 μm.

Mentions: Staining of LBCs with both Sat3 or Sat4 antisera showed an extraordinarily prominent signal on a single set of loops located on bivalent no. 3. In addition, there was moderate staining of all other loops. Preimmune sera, in contrast, showed no chromosomal staining indicating that the signals were specific for xlADAR1 (Fig. 2, and data not shown). Staining with Sat3 showed a weak background on C snurposomes that was also observed in the corresponding preimmune serum whereas staining with Sat4 showed a weak background on nucleoli also observable in the corresponding preimmune serum. Those weak background signals that were only observed at low dilutions of antisera were thus considered as nonspecific background signals. The staining of the brilliant loop was so intense that it could still be detected at antisera dilutions up to 1:3,000. In contrast, for the majority of all other loops staining was well visible at antisera dilutions of 1:500. Thus, it was hard to take photographic pictures with both the brilliant loop and the regular loops at good resolutions. Therefore, Fig. 2 shows images of bivalent no. 3 including the brilliantly labeling loop and images of other bivalents, showing the label observed on all other 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)

xlADAR1 localizes to the nascent RNP matrix and is specifically enriched on a special loop on  bivalent no. 3. LBCs were  prepared from oocytes  expressing myc-tagged  ADAR1.1. (a–d) Normal  LBC and (e–f) bivalent no. 3.  (a and e) DIC image, (b and  f) DAPI staining, (c and g)  localization of endogenous  ADAR1 detected with SAT4  antiserum in the rhodamine  channel and (d and h) localization of myc-tagged  ADAR1.1 detected with  mAb 9E10 in the fluorescein  channel. Endogenous and ectopically expressed ADAR1  is localized to LBC loops. (g  and h) A special loop on  bivalent no. 3 is enriched for  endogenous (g) and ectopically expressed ADAR1 (h).  The special loop is marked  by arrows. Faint background  signals can be seen on nucleoli (N) or snurposomes (S)  by staining with SAT4 antiserum that is not seen with  myc-tagged ADAR1.1. Bar,  10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132932&req=5

Figure 2: xlADAR1 localizes to the nascent RNP matrix and is specifically enriched on a special loop on bivalent no. 3. LBCs were prepared from oocytes expressing myc-tagged ADAR1.1. (a–d) Normal LBC and (e–f) bivalent no. 3. (a and e) DIC image, (b and f) DAPI staining, (c and g) localization of endogenous ADAR1 detected with SAT4 antiserum in the rhodamine channel and (d and h) localization of myc-tagged ADAR1.1 detected with mAb 9E10 in the fluorescein channel. Endogenous and ectopically expressed ADAR1 is localized to LBC loops. (g and h) A special loop on bivalent no. 3 is enriched for endogenous (g) and ectopically expressed ADAR1 (h). The special loop is marked by arrows. Faint background signals can be seen on nucleoli (N) or snurposomes (S) by staining with SAT4 antiserum that is not seen with myc-tagged ADAR1.1. Bar, 10 μm.
Mentions: Staining of LBCs with both Sat3 or Sat4 antisera showed an extraordinarily prominent signal on a single set of loops located on bivalent no. 3. In addition, there was moderate staining of all other loops. Preimmune sera, in contrast, showed no chromosomal staining indicating that the signals were specific for xlADAR1 (Fig. 2, and data not shown). Staining with Sat3 showed a weak background on C snurposomes that was also observed in the corresponding preimmune serum whereas staining with Sat4 showed a weak background on nucleoli also observable in the corresponding preimmune serum. Those weak background signals that were only observed at low dilutions of antisera were thus considered as nonspecific background signals. The staining of the brilliant loop was so intense that it could still be detected at antisera dilutions up to 1:3,000. In contrast, for the majority of all other loops staining was well visible at antisera dilutions of 1:500. Thus, it was hard to take photographic pictures with both the brilliant loop and the regular loops at good resolutions. Therefore, Fig. 2 shows images of bivalent no. 3 including the brilliantly labeling loop and images of other bivalents, showing the label observed on all other 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
Related in: MedlinePlus