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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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Splicing is not required for ADAR1 localization. (a and d) DIC images,  (b and e) DAPI staining, and  (c and f) ADAR1 localization detected by staining with  SAT4 antiserum in the fluorescein channel. (a–c) Staining of bivalent no. 3 from an  untreated oocyte shows localization of ADAR1 on the  RNP matrix of regular loops  and on the special loop. (d–f)  24 h after injection of the  U2b oligo transcription has  resumed giving rise to prominent loops (d). (f) Staining  with SAT4 antiserum shows  the presence of ADAR1 on  the loop matrix and on the  special loop on bivalent no. 3.  Bar, 10 μm.
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Figure 5: Splicing is not required for ADAR1 localization. (a and d) DIC images, (b and e) DAPI staining, and (c and f) ADAR1 localization detected by staining with SAT4 antiserum in the fluorescein channel. (a–c) Staining of bivalent no. 3 from an untreated oocyte shows localization of ADAR1 on the RNP matrix of regular loops and on the special loop. (d–f) 24 h after injection of the U2b oligo transcription has resumed giving rise to prominent loops (d). (f) Staining with SAT4 antiserum shows the presence of ADAR1 on the loop matrix and on the special loop on bivalent no. 3. Bar, 10 μm.

Mentions: Destruction of U2 was monitored by Northern blotting of RNAs isolated from individual GVs of injected and control oocytes (data not shown). After transcription resumed, LBCs were tested for the localization of ADAR1 by staining with Sat3 antiserum (Fig. 5). Interestingly, no difference in ADAR1 localization could be observed in U2 depleted oocytes, indicating that splicing and spliceosome formation is not required for the association of ADAR1 with the nascent RNP matrix.


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)

Splicing is not required for ADAR1 localization. (a and d) DIC images,  (b and e) DAPI staining, and  (c and f) ADAR1 localization detected by staining with  SAT4 antiserum in the fluorescein channel. (a–c) Staining of bivalent no. 3 from an  untreated oocyte shows localization of ADAR1 on the  RNP matrix of regular loops  and on the special loop. (d–f)  24 h after injection of the  U2b oligo transcription has  resumed giving rise to prominent loops (d). (f) Staining  with SAT4 antiserum shows  the presence of ADAR1 on  the loop matrix and on the  special loop on bivalent no. 3.  Bar, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132932&req=5

Figure 5: Splicing is not required for ADAR1 localization. (a and d) DIC images, (b and e) DAPI staining, and (c and f) ADAR1 localization detected by staining with SAT4 antiserum in the fluorescein channel. (a–c) Staining of bivalent no. 3 from an untreated oocyte shows localization of ADAR1 on the RNP matrix of regular loops and on the special loop. (d–f) 24 h after injection of the U2b oligo transcription has resumed giving rise to prominent loops (d). (f) Staining with SAT4 antiserum shows the presence of ADAR1 on the loop matrix and on the special loop on bivalent no. 3. Bar, 10 μm.
Mentions: Destruction of U2 was monitored by Northern blotting of RNAs isolated from individual GVs of injected and control oocytes (data not shown). After transcription resumed, LBCs were tested for the localization of ADAR1 by staining with Sat3 antiserum (Fig. 5). Interestingly, no difference in ADAR1 localization could be observed in U2 depleted oocytes, indicating that splicing and spliceosome formation is not required for the association of ADAR1 with the nascent RNP matrix.

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