Deamination of mammalian glutamate receptor RNA by Xenopus dsRNA adenosine deaminase: similarities to in vivo RNA editing.

Double-stranded RNA (dsRNA) adenosine deaminase (dsRAD) converts adenosines to inosines within dsRNA. A great deal of evidence suggests that dsRAD or a related enzyme edits mammalian glutamate receptor mRNA in vivo. Here we map the deamination sites that occur in a truncated glutamate receptor-B (gluR-B) mRNA after incubation with pure Xenopus dsRAD. We find remarkable similarities, as well as distinct differences, between the observed deamination sites and the sites reported to be edited within RNAs isolated from mammalian brain. For example, although deamination at the biologically relevant Q/R editing site occurs, it occurs much less frequently than editing at this site in vivo. We hypothesize that the similarities between the deamination and editing patterns exist because the deamination specificity that is intrinsic to dsRAD is involved in selecting editing sites in vivo. We propose that the observed differences are due to the absence of accessory factors that play indirect roles in vivo, such as binding to and occluding certain sites from dsRAD, or promoting the RNA structure required for correct and efficient editing. The work reported here also suggests that dsRAD is capable of much more selectivity than previously thought; a minimal number of deamination sites (average < or = 5) were found in each gluR-B RNA. We speculate that the observed selectivity is due to the various structural elements (mismatches, bulges, loops) that periodically interrupt the base paired region required for editing.

Binding properties of newly identified Xenopus proteins containing dsRNA-binding motifs.

BACKGROUND: Although most RNA-binding proteins recognize a complex set of structural motifs in their RNA target, the double-stranded (ds) RNA-binding proteins are limited to interactions with double helices. Recently, it has been discovered that some dsRNA-binding proteins share regions of amino-acid similarity known as dsRNA-binding motifs. RESULTS: A Xenopus ovary cDNA expression library was screened with radiolabeled dsRNA to identify previously uncharacterized dsRNA-binding proteins. The analysis of an incomplete cDNA identified during the screen led to the discovery of two longer cDNAs of related sequence. The proteins encoded by these cDNAs each contained two dsRNA-binding motifs, in glycine. The nucleic-acid-binding properties of a fusion protein containing the two dsRNA-binding motifs and the auxiliary domain were analyzed using a gel mobility shift assay. The fusion protein bound dsRNA of a variety of different sequences, and exhibited a preference for binding to dsRNA and RNA-DNA hybrids over other nucleic acids. Appropriate mRNAs, corresponding to each cDNA, were detected in polyadenylated RNA isolated from Xenopus stage VI oocytes, but translation of one of the mRNAs appeared to be masked until meiotic maturation. CONCLUSION: dsRNA-binding motifs are often found in proteins that bind dsRNA, and our results show that they can be associated with auxiliary domains rich in arginine and glycine. These motifs can confer very tight binding to dsRNA. Binding can also occur to RNA-DNA hybrids, suggesting recognition of some aspect of the A-form helical structure that is adopted by both dsRNA and RNA-DNA hybrids.

Ionic requirements for establishment of an embryonic axis in Pelvetia zygotes.

Previous reports have shown that fertilized Pelvetia fastigiata (J. Ag.) DeToni eggs generate a transcellular ionic current which correlates temporally and spatially with establishment of a rhizoid/thallus axis. In order to understand more fully the ionic controls of development, we have determined the ionic requirements for axis formation induced by unilateral light. Formation of the developmental axis was independent of the presence of individual ions in artificial seawater. This finding indicates that transcellular circulation of a particular ion is not obligatory for polarization, and it confirms earlier work showing that calcium circulation is not fundamental to axis establishment in Fucus zygotes. Polarization was not, however, completely independent of ionic conditions; zygotes were unable to form an axis in pure sucrose solutions. Single salts were added to sucrose to determine which ions were sufficient to permit polarization. Salts of impermeant monovalent cations and salts of divalent cations supported polarization weakly or not at all. By contrast, zygotes photopolarized well in KCl, and other alkali metals substituted for K(+) with varying effectiveness (Rb(+)>Na(+)> Cs(+)≫ Li(+)). The anion was unimportant; a variety of different potassium salts all supported polarization equally well. The mechanism by which KCl promotes polarization is not yet understood, but may involve transport through K(+) channels.