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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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Double staining of  the special loop on bivalent  no. 3 with various antibodies  and SAT antiserum. (a, d,  g, and j) DIC images, (b, e,  h, and k) fluorescein channel,  and (c, f, i, and l) staining  with SAT4 antiserum in the  rhodamine channel. Arrows  mark the position of the special loop. (a–c) Staining with  mAbH14 (b) shows the presence of RNA Pol-II on all  regular loops as a fine signal  seen in the center of each  loop. However, Pol-II is absent from the special loop  which is brilliantly labeled by  SAT4 antiserum (c). (d–f)  Staining with mAb K121 indicates the presence of 3mG  snRNP cap structures on the  special loop (e) which is also  labeled with SAT4 antiserum (f). (g–i) mAb Y12 stains  regular loops and the special  loop indicating the presence  of Sm proteins on the special  loop (h). (j–l) The SR splicing  factor SC35 can also be found  on the special loop (k). Bar,  10 μm.
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Figure 4: Double staining of the special loop on bivalent no. 3 with various antibodies and SAT antiserum. (a, d, g, and j) DIC images, (b, e, h, and k) fluorescein channel, and (c, f, i, and l) staining with SAT4 antiserum in the rhodamine channel. Arrows mark the position of the special loop. (a–c) Staining with mAbH14 (b) shows the presence of RNA Pol-II on all regular loops as a fine signal seen in the center of each loop. However, Pol-II is absent from the special loop which is brilliantly labeled by SAT4 antiserum (c). (d–f) Staining with mAb K121 indicates the presence of 3mG snRNP cap structures on the special loop (e) which is also labeled with SAT4 antiserum (f). (g–i) mAb Y12 stains regular loops and the special loop indicating the presence of Sm proteins on the special loop (h). (j–l) The SR splicing factor SC35 can also be found on the special loop (k). Bar, 10 μm.

Mentions: To test the former possibility we performed double immunofluorescence staining with mAbs H14 or CC3, both directed against Pol-II, and Sat4 antiserum (Vincent et al., 1996; Kim et al., 1997). This data showed the presence of Pol-II on all regular loops, as a faint line of signal could be seen throughout the axis of loops. The brilliantly labeling loops, in contrast, showed no detectable signal with either mAb directed against Pol-II, suggesting that no Pol-II-dependent transcription occurs at this particular loop (Fig. 4).


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)

Double staining of  the special loop on bivalent  no. 3 with various antibodies  and SAT antiserum. (a, d,  g, and j) DIC images, (b, e,  h, and k) fluorescein channel,  and (c, f, i, and l) staining  with SAT4 antiserum in the  rhodamine channel. Arrows  mark the position of the special loop. (a–c) Staining with  mAbH14 (b) shows the presence of RNA Pol-II on all  regular loops as a fine signal  seen in the center of each  loop. However, Pol-II is absent from the special loop  which is brilliantly labeled by  SAT4 antiserum (c). (d–f)  Staining with mAb K121 indicates the presence of 3mG  snRNP cap structures on the  special loop (e) which is also  labeled with SAT4 antiserum (f). (g–i) mAb Y12 stains  regular loops and the special  loop indicating the presence  of Sm proteins on the special  loop (h). (j–l) The SR splicing  factor SC35 can also be found  on the special loop (k). Bar,  10 μm.
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Related In: Results  -  Collection

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Figure 4: Double staining of the special loop on bivalent no. 3 with various antibodies and SAT antiserum. (a, d, g, and j) DIC images, (b, e, h, and k) fluorescein channel, and (c, f, i, and l) staining with SAT4 antiserum in the rhodamine channel. Arrows mark the position of the special loop. (a–c) Staining with mAbH14 (b) shows the presence of RNA Pol-II on all regular loops as a fine signal seen in the center of each loop. However, Pol-II is absent from the special loop which is brilliantly labeled by SAT4 antiserum (c). (d–f) Staining with mAb K121 indicates the presence of 3mG snRNP cap structures on the special loop (e) which is also labeled with SAT4 antiserum (f). (g–i) mAb Y12 stains regular loops and the special loop indicating the presence of Sm proteins on the special loop (h). (j–l) The SR splicing factor SC35 can also be found on the special loop (k). Bar, 10 μm.
Mentions: To test the former possibility we performed double immunofluorescence staining with mAbs H14 or CC3, both directed against Pol-II, and Sat4 antiserum (Vincent et al., 1996; Kim et al., 1997). This data showed the presence of Pol-II on all regular loops, as a faint line of signal could be seen throughout the axis of loops. The brilliantly labeling loops, in contrast, showed no detectable signal with either mAb directed against Pol-II, suggesting that no Pol-II-dependent transcription occurs at this particular loop (Fig. 4).

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