Sallacz NB and Jantsch MF (2005), Chromosomal storage of the RNA-editing enzyme A...

XB-ART-34620

Mol Biol Cell 2005 Jul 01;167:3377-86. doi: 10.1242/dev.01814.

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Chromosomal storage of the RNA-editing enzyme ADAR1 in Xenopus oocytes.

Sallacz NB , Jantsch MF .

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ADARs (adenosine deaminases that act on RNA) are RNA-editing enzymes that convert adenosines to inosines in structured or double-stranded RNAs. Expression and intracellular distribution of ADAR1 is controlled by a plethora of mechanisms suggesting that enzyme activity has to be tightly regulated. Mammalian ADAR1 is a shuttling protein, whereas Xenopus ADAR1 is exclusively nuclear. In oocytes, Xenopus ADAR1 associates with most nascent transcripts but is strongly enriched at a specific site on chromosome 3, termed the special loop. Enrichment at this site requires the presence of RNAs but is independent of ongoing transcription. Here we show that RNAs transcribed elsewhere in the genome accumulate at the special loop even in the absence of transcription. In situ hybridization experiments, however, indicate the absence of known editing substrates from this site. In the absence of transcription also other RNA binding and processing factors accumulate at the special loop, suggesting that ADAR1 is stored or assembled at the special loop in an RNA-containing complex. Nuclear injection of RNAs providing binding sites for ADAR1 dissociates the enzyme from the special loop, supporting the notion that the special loop represents a site where ADAR1 is stored, possibly for later use during development.

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Species referenced: Xenopus laevis
Genes referenced: adar fgf2 fubp1 hnrnpc srsf2

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	Figure 1. RNA turnover at the special loop. To detect newly synthesized RNA, oocytes were injected with brUTP. At several time points after injection, chromosome spreads were stained with anti-ADAR1 (ADAR1) or brUTP (brUTP) antibodies, respectively. Nomarski optics image of the same region (NOM). Arrowheads mark the position of the special loop on bivalent 3. Six hours (6h) after brUTP injection the majority of regular loops shows massive incorporation of brUTP, whereas the special loop highlighted with the ADAR1 antibody shows no brUTP. Forty-eight hours (48 h) after brUTP injection the special loop appears unlabeled when compared with regular loops. (8d AMD) Eight days after injection followed by 8 h of AMD treatment transcription at regular loops has ceased, allowing the detection of brUTP-labeled RNAs at the special loop. Scale bar, 10 m
	Figure 2. Fluorescence in situ hybridization (FISH) reveals the absence of bFGF transcripts from the special loop. bFGF was detected by FISH with a fluorescein-labeled antisense probe (FISH bFGF), and the special loop was detected by subsequent immunostaining with anti-ADAR1 antiserum (xlADAR1). bFGF RNA could be consistently detected on three different chromosomes (arrows, top three rows), which were not further identified, whereas bFGF RNA was absent from the special loop (arrowhead, bottom row). The faint signals detected in the anti-ADAR immunostaining at the positions corresponding to the FISH signals (arrows) result from the cross-reactivity of the antibody-based signal amplification system used for FISH. Chromosomal images stained with DAPI and visualized by Nomarski optics are shown for comparison. Scale bar, 10 m.
	Figure 3. RNA processing factors accumulate at the special loop in the absence of transcription. Chromosomal spreads were stained with antibodies against the shuttling hnRNP A/B, the nonshuttling hnRNP L, the nonshuttling SR-protein SC35, or the trimethyl G (3mG) cap found on most splicing snRNPs, both in the presence and absence (AMD) of transcription. xlADAR1 remains at the special loop after AMD treatment (arrowheads), whereas regular loops are stripped of their RNP matrix and fuse with the chromosomal axis (see Nomarski image of AMD-treated chromosomes). HnRNPs A/B and L are absent from the special loop under normal conditions but show strong (A/B) and moderate (L) enrichment at this site upon AMD treatment. SC35, in contrast, is present but not enriched at the special loop under normal conditions but strongly accumulates there when transcription is inhibited. 3mG, indicative of snRNPs, is always enriched at the special loop and thus behaves like ADAR1. Scale bar, 10 m
	Figure 4. Human ADAR1 is a shuttling protein, whereas Xenopus ADAR1 is not. Heterokaryons of Xenopus XlA6 and HeLa cells were produced by PEG fusion. The HeLa nuclei were identified by distamycin ADAPI (DA/DAPI) staining, which results in DAPI-bright heterochromatin spots, whereas the Xenopus nuclei are stained homogeneously. Species-specific antibodies against human ADAR1 (top row) and Xenopus ADAR1 (bottom row) were used to detect the respective proteins in the heterokaryons. Human ADAR1 shuttles out of the human nucleus and can be found in the Xenopus nucleus (hsADAR1), whereas Xenopus ADAR1 remains in the Xenopus nucleus (xlADAR1). Whole cells are visualized by Nomarski optics (NOM). Scale bar, 10 m.
	Figure 5. Xenopus ADAR1 can be stripped off the special loop by excess substrate. Injection of double-stranded RNA (dsRNA 2/3) into Xenopus oocyte nuclei preferentially removes ADAR1 from the special loop (arrowheads), whereas injection of single-stranded RNA (ssRNA 3) has no effect on ADAR1 localization. The effect is most prominent 3 h after injection of the oligo. Twenty-four hours after injection ADAR1 redecorates the special loop normally. Bar, 10 m.
	Figure 6. The special loop, a chromosomal storage site? RNAs transcribed somewhere in the genome (A) move to the special loop on chromosome 3 where they accumulate (B). RNAs accumulated at the special loop define this site as a storage region for excess RNA-processing proteins and serve as a binding platform to sequester proteins to this site (C).

References [+] :

Athanasiadis, Widespread A-to-I RNA editing of Alu-containing mRNAs in the human transcriptome. 2004, Pubmed