Double-stranded RNA unwinding and modifying activity is detected ubiquitously in primary tissues and cell lines.

Wagner, Richard W.; Yoo, C J; Wrabetz, Lawrence; Kamholz, John; Buchhalter, J; Hassan, Nassef F.; Khalili, Kamel; Kim, S U; Perussia, Bice; McMorris, F. Arthur

doi:10.1128/mcb.10.10.5586

Cited by 93 publications

(61 citation statements)

References 24 publications

(26 reference statements)

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“…Even though this probe contained the sequence for the first dsRNA binding domain, it did not cross-hybridize with other mRNAs encoding dsRNA binding domains. The mRNA for the dsRNA adenosine deaminase was present in all tissues examined, in agreement with earlier reports that demonstrated a widespread expression of the deaminase (45). Northern blot analysis of poly(A) ϩ RNA from calf thymus with the partial bovine cDNA clone as a probe also showed a band of Ͼ7.5 kb (data not shown).…”

Section: Northern Blot Analysis Northern Blot Analysis Of Human Tisssupporting

confidence: 77%

Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase

O’Connell

Krause

Higuchi

et al. 1995

Molecular and Cellular Biology

248

231

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Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNAspecific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain. Double-stranded RNA (dsRNA)-specific adenosine deaminase is a mammalian RNA-modifying enzyme that was discovered upon performing antisense experiments with Xenopus laevis (2, 36). The enzyme destabilizes duplex RNA by converting adenosine to inosine by hydrolytic deamination (34), which results in unstable U ⅐ I base pairs so that the dsRNA becomes unwound. While in vitro there is no sequence specificity for substrate RNAs, the activity is specific for dsRNAs, and a minimum of 100 bp of either intermolecular or intramolecular duplex seems to be required for efficient modification (30).The biological function of the enzyme as well as the full range of its physiological substrate RNAs are not known. The deaminase has been implicated in editing the RNAs of certain subunits of glutamate-gated ion channel receptors in the brain, because of the dependence of this editing event on intramolecular RNA ⅐ RNA base pairing (19,24,37). The dsRNA adenosine deaminase appears also to be responsible for the generation of defective measles virus with biased hypermutations that result in a lethal central nervous system disease, measles inclusion body encephalitis (4).This enzyme is of interest not only because of its assumed biological functions but also because its mechanism of action is intriguing. dsRNA differs from duplex DNA in forming an A-type helix. In A-form RNA, the minor groove is shallow and wide (10 to 11 Å [1 Å ϭ 0.1 nm]) and the major groove is deep and narrow (3.5 Å) (38). RNA-binding proteins usually bind to the major groove at the end of a helix or at a bulge in RNA structures where the major groove is more accessible (38). The binding of human immunodeficiency virus Tat protein to its target RNA TAR is an example, as the binding occurs on the major groove in the region of a bulge (35,47,48). The dsRNA adenosine deaminase binds to continuous duplex RNA; therefore, it must be able to recognize and access the adenosines that it deaminates even though this amino group is at position 6 of the base and lies in the major groove. This position is inacc...

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Section: Northern Blot Analysis Northern Blot Analysis Of Human Tisssupporting

confidence: 77%

Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase

O’Connell

Krause

Higuchi

et al. 1995

Molecular and Cellular Biology

248

231

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“…Further experiments revealed that dsRAD was present in post-MBT embryos but was localized to the nucleus and thus not detected in early experiments involving cytoplasmic microinjections (5). dsRAD has also been shown to be nuclear in the somatic cells of mammals (34). Taken together, these results raised the possibility that dsRAD is in the nuclei of stage VI oocytes and exists in the cytoplasm of eggs and early embryos because it is released from the nucleus during meiotic maturation, the process by which a stage VI oocyte becomes an egg (for a review, see reference 31).…”

mentioning

confidence: 99%

The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification.

Saccomanno¹,

Bass²

1994

Mol. Cell. Biol.

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Here we describe studies of double-stranded RNA (dsRNA) adenosine deaminase in Xenopus laevis, in particular during meiotic maturation, the period during which a stage VI oocyte matures to an egg. We show that dsRNA adenosine deaminase is in the nuclei of stage VI oocytes. Most importantly, we demonstrate that the cytoplasm of stage VI oocytes contains a factor that protects microinjected dsRNA from deamination when dsRNA adenosine deaminase is released from the nucleus during meiotic maturation. Our data suggest that the protection factor is a cytoplasmic dsRNA-binding protein or proteins that bind to dsRNA in a sequence-independent manner to occlude dsRNA from binding to dsRNA adenosine deaminase. The cytoplasmic double-stranded RNA-binding protein(s) does not bind to other nucleic acids and can be titrated at high concentrations of dsRNA. These studies raise the question of whether all dsRNA-binding proteins share endogenous substrates and also suggest potential means of regulating dsRNA adenosine deaminase in vivo.The double-stranded RNA (dsRNA) adenosine deaminase (dsRAD), initially called the unwinding/modifying activity, was detected first in Xenopus laevis (4, 25) and subsequently in organisms throughout the animal kingdom (34; for reviews, see references 1, 2, and 17). This enzyme deaminates adenosines within dsRNA to produce inosines (24). Recently dsRAD has been purified from Xenopus eggs; this protein is a single subunit that migrates at 120 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (14). In vitro studies demonstrate that dsRAD can deaminate adenosines within intermolecular and intramolecular RNA duplexes (21, 29) but cannot act on adenosines within DNA or single-stranded RNA (ssRNA) (4, 33). Strong evidence indicating that dsRAD is responsible for RNA editing of mRNAs encoding the B subunit of a specific type of L-glutamate-activated ion channel has been presented (13). In addition to RNA editing, other biological roles for dsRAD have been proposed (for a review, see reference 2), and it is possible that dsRAD plays multiple roles in vivo.Initial experiments in X laevis showed that dsRAD could easily be detected by assaying the fate of dsRNA microinjected into the cytoplasm of eggs and early embryos (.8 cells; 4, 25).However, the activity could not be observed in similar microinjections into the cytoplasm of oocytes or embryos at later stages of development, specifically, subsequent to the midblastula transition (post-MBT). Further experiments revealed that dsRAD was present in post-MBT embryos but was localized to the nucleus and thus not detected in early experiments involving cytoplasmic microinjections (5). dsRAD has also been shown to be nuclear in the somatic cells of mammals (34). Taken together, these results raised the possibility that dsRAD is in the nuclei of stage VI oocytes and exists in the cytoplasm of eggs and early embryos because it is released from the nucleus during meiotic maturation, the process by which a stage VI oocyte becomes an egg (for a review, see ...

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“…Although the level of ADAR1 transcript is increased by treatment with both IFN-␣ and IFN-␥ (9, 13, 17), a significant basal level of ADAR1 transcript is observed in human cell lines and organs in the absence of IFN treatment (9)(10)(11). Because of the important role that ADAR1 plays in the editing of cellular neurotransmitter receptor pre-mRNAs in the apparent absence of IFN treatment (2,18) coupled with the unresolved origin of the constitutively expressed Ϸ110-kDa form of ADAR1 protein ubiquitously observed at high levels in the nucleus of animal cells in the absence of IFN treatment (9,20), and because of the potential role of the IFN-inducible Ϸ150-kDa form of ADAR1 in the antiviral actions of IFN (9, 21), we have attempted to define the elements responsible for ADAR1 transcriptional control. As a step toward this goal, we isolated and characterized genomic clones of the human ADAR1 gene (14,15).…”

mentioning

confidence: 99%

Human RNA-specific adenosine deaminase ADAR1 transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible

George

Samuel

1999

Proc. Natl. Acad. Sci. U.S.A.

278

253

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Double-stranded RNA unwinding and modifying activity is detected ubiquitously in primary tissues and cell lines.

Cited by 93 publications

References 24 publications

Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase

Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase

The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification.

Human RNA-specific adenosine deaminase ADAR1 transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible

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