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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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Localization of  ADAR1 to LBC loops is  RNA dependent. Staining of  bivalent no. 3 with SAT4 antiserum. (a, d, g, j, and m)  DIC image, (b, e, h, k, and n)  DAPI staining, and (c, f, i, l,  and o) staining with Sat4  in the fluorescein channel.  (a–c) ADAR1 is localized to  LBC loops and is specifically  enriched on a special loop.  (d–f) Staining with Sat4 can  be blocked by ADAR1 peptide. (f) SAT signal is almost  completely diminished when  the antiserum was blocked  with ADAR1 peptide originally used for the immunization. (g–i) SAT staining is  sensitive to RNAse treatment. LBCs were digested  with RNAse before staining  with Sat4 antiserum. (g) The  loops appear “stripped” after  RNAse treatment, and (i) no  signal can be observed in the  fluorescein channel. (j–l)  Treatment with actinomycin  D inhibits transcription and  diminishes staining of regular  loops but not of the special  loop on bivalent no. 3. (j)  DIC image of bivalent no. 3  prepared from an oocyte after incubation in AMD. The  chromosomal axes is condensed and shows no transcriptionally active loops.  Also nucleoli (N) change  their morphology. (k) The  condensed, shortened chromosomal axes is well stained  with DAPI. The position of the special loop forming a “double loop bridge” is seen by the interrupted DAPI staining of the chromosomal axes on both homologues (arrowhead). (l) As transcription is inhibited no ADAR1 staining can be observed. Only the special  loop is still brilliantly labeled by SAT4 antiserum. (m–o) Injection of an unrelated oligonucleotide temporarily inhibits transcription but  does not affect ADAR1 localization on the special loop on bivalent no. 3. (m) DIC image of bivalent no. 3 prepared from an oocyte  24 h after injection of an oligonucleotide. The presence of loops on the chromosome indicates that transcription has already resumed.  (n) DAPI image of the same region. (o) ADAR1 can be detected on most transcripts and on the special loop by staining with SAT4 antiserum. Note: Shortly after injection of the oligo transcription seizes and no ADAR1 staining can be detected on regular loops (not  shown). However, staining of the brilliantly labeling loop is not affected by this treatment. Bars, 10 μm.
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Figure 3: Localization of ADAR1 to LBC loops is RNA dependent. Staining of bivalent no. 3 with SAT4 antiserum. (a, d, g, j, and m) DIC image, (b, e, h, k, and n) DAPI staining, and (c, f, i, l, and o) staining with Sat4 in the fluorescein channel. (a–c) ADAR1 is localized to LBC loops and is specifically enriched on a special loop. (d–f) Staining with Sat4 can be blocked by ADAR1 peptide. (f) SAT signal is almost completely diminished when the antiserum was blocked with ADAR1 peptide originally used for the immunization. (g–i) SAT staining is sensitive to RNAse treatment. LBCs were digested with RNAse before staining with Sat4 antiserum. (g) The loops appear “stripped” after RNAse treatment, and (i) no signal can be observed in the fluorescein channel. (j–l) Treatment with actinomycin D inhibits transcription and diminishes staining of regular loops but not of the special loop on bivalent no. 3. (j) DIC image of bivalent no. 3 prepared from an oocyte after incubation in AMD. The chromosomal axes is condensed and shows no transcriptionally active loops. Also nucleoli (N) change their morphology. (k) The condensed, shortened chromosomal axes is well stained with DAPI. The position of the special loop forming a “double loop bridge” is seen by the interrupted DAPI staining of the chromosomal axes on both homologues (arrowhead). (l) As transcription is inhibited no ADAR1 staining can be observed. Only the special loop is still brilliantly labeled by SAT4 antiserum. (m–o) Injection of an unrelated oligonucleotide temporarily inhibits transcription but does not affect ADAR1 localization on the special loop on bivalent no. 3. (m) DIC image of bivalent no. 3 prepared from an oocyte 24 h after injection of an oligonucleotide. The presence of loops on the chromosome indicates that transcription has already resumed. (n) DAPI image of the same region. (o) ADAR1 can be detected on most transcripts and on the special loop by staining with SAT4 antiserum. Note: Shortly after injection of the oligo transcription seizes and no ADAR1 staining can be detected on regular loops (not shown). However, staining of the brilliantly labeling loop is not affected by this treatment. Bars, 10 μm.

Mentions: As a further control, to show that the chromosomal staining with both Sat3 and Sat4 antisera was specific for ADAR1, we blocked both antisera with the fusion proteins used to generate antibodies. This blocking eliminated chromosomal staining almost completely, leaving only a faint signal on the intensely labeling loops on bivalent no. 3. Thus, the observed signals reflect the localization of endogenous ADAR1 (Fig. 3).


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)

Localization of  ADAR1 to LBC loops is  RNA dependent. Staining of  bivalent no. 3 with SAT4 antiserum. (a, d, g, j, and m)  DIC image, (b, e, h, k, and n)  DAPI staining, and (c, f, i, l,  and o) staining with Sat4  in the fluorescein channel.  (a–c) ADAR1 is localized to  LBC loops and is specifically  enriched on a special loop.  (d–f) Staining with Sat4 can  be blocked by ADAR1 peptide. (f) SAT signal is almost  completely diminished when  the antiserum was blocked  with ADAR1 peptide originally used for the immunization. (g–i) SAT staining is  sensitive to RNAse treatment. LBCs were digested  with RNAse before staining  with Sat4 antiserum. (g) The  loops appear “stripped” after  RNAse treatment, and (i) no  signal can be observed in the  fluorescein channel. (j–l)  Treatment with actinomycin  D inhibits transcription and  diminishes staining of regular  loops but not of the special  loop on bivalent no. 3. (j)  DIC image of bivalent no. 3  prepared from an oocyte after incubation in AMD. The  chromosomal axes is condensed and shows no transcriptionally active loops.  Also nucleoli (N) change  their morphology. (k) The  condensed, shortened chromosomal axes is well stained  with DAPI. The position of the special loop forming a “double loop bridge” is seen by the interrupted DAPI staining of the chromosomal axes on both homologues (arrowhead). (l) As transcription is inhibited no ADAR1 staining can be observed. Only the special  loop is still brilliantly labeled by SAT4 antiserum. (m–o) Injection of an unrelated oligonucleotide temporarily inhibits transcription but  does not affect ADAR1 localization on the special loop on bivalent no. 3. (m) DIC image of bivalent no. 3 prepared from an oocyte  24 h after injection of an oligonucleotide. The presence of loops on the chromosome indicates that transcription has already resumed.  (n) DAPI image of the same region. (o) ADAR1 can be detected on most transcripts and on the special loop by staining with SAT4 antiserum. Note: Shortly after injection of the oligo transcription seizes and no ADAR1 staining can be detected on regular loops (not  shown). However, staining of the brilliantly labeling loop is not affected by this treatment. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Localization of ADAR1 to LBC loops is RNA dependent. Staining of bivalent no. 3 with SAT4 antiserum. (a, d, g, j, and m) DIC image, (b, e, h, k, and n) DAPI staining, and (c, f, i, l, and o) staining with Sat4 in the fluorescein channel. (a–c) ADAR1 is localized to LBC loops and is specifically enriched on a special loop. (d–f) Staining with Sat4 can be blocked by ADAR1 peptide. (f) SAT signal is almost completely diminished when the antiserum was blocked with ADAR1 peptide originally used for the immunization. (g–i) SAT staining is sensitive to RNAse treatment. LBCs were digested with RNAse before staining with Sat4 antiserum. (g) The loops appear “stripped” after RNAse treatment, and (i) no signal can be observed in the fluorescein channel. (j–l) Treatment with actinomycin D inhibits transcription and diminishes staining of regular loops but not of the special loop on bivalent no. 3. (j) DIC image of bivalent no. 3 prepared from an oocyte after incubation in AMD. The chromosomal axes is condensed and shows no transcriptionally active loops. Also nucleoli (N) change their morphology. (k) The condensed, shortened chromosomal axes is well stained with DAPI. The position of the special loop forming a “double loop bridge” is seen by the interrupted DAPI staining of the chromosomal axes on both homologues (arrowhead). (l) As transcription is inhibited no ADAR1 staining can be observed. Only the special loop is still brilliantly labeled by SAT4 antiserum. (m–o) Injection of an unrelated oligonucleotide temporarily inhibits transcription but does not affect ADAR1 localization on the special loop on bivalent no. 3. (m) DIC image of bivalent no. 3 prepared from an oocyte 24 h after injection of an oligonucleotide. The presence of loops on the chromosome indicates that transcription has already resumed. (n) DAPI image of the same region. (o) ADAR1 can be detected on most transcripts and on the special loop by staining with SAT4 antiserum. Note: Shortly after injection of the oligo transcription seizes and no ADAR1 staining can be detected on regular loops (not shown). However, staining of the brilliantly labeling loop is not affected by this treatment. Bars, 10 μm.
Mentions: As a further control, to show that the chromosomal staining with both Sat3 and Sat4 antisera was specific for ADAR1, we blocked both antisera with the fusion proteins used to generate antibodies. This blocking eliminated chromosomal staining almost completely, leaving only a faint signal on the intensely labeling loops on bivalent no. 3. Thus, the observed signals reflect the localization of endogenous ADAR1 (Fig. 3).

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