Here we take advantage of the well-characterized and simple nervous system of Caenorhabditis elegans to further our understanding of the functions of RNA editing. We describe the two C.elegans ADAR genes, adr-1 and adr-2, and characterize strains containing homozygous deletions in each, or both, of these genes. We find that adr-1 is expressed in most, if not all, cells of the C.elegans nervous system and also in the developing vulva. Using chemotaxis assays, we show that both ADARs are important for normal behavior. Biochemical, molecular and phenotypic analyses indicate that ADR-1 and ADR-2 have distinct roles in C.elegans, but sometimes act together.

The signal transduction and activation of RNA (STAR) family of RNA-binding proteins, whose members are evolutionarily conserved from yeast to humans, are important for a number of developmental decisions. For example, in the mouse, quaking proteins (QKI-5, QKI-6, and QKI-7) are essential for embryogenesis and myelination , whereas a closely related protein in Caenorhabditis elegans, germline defective-1 (GLD-1), is necessary for germline development. Recently, GLD-1 was found to be a translational repressor that acts through regulatory elements, called TGEs (for tra-2 and GLI elements), present in the 3 untranslated region of the sex-determining gene tra-2. This gene promotes female development, and repression of tra-2 translation by TGEs is necessary for the male cell fates. The finding that GLD-1 inhibits tra-2 translation raises the possibility that other STAR family members act by a similar mechanism to control gene activity. Here we demonstrate, both in vitro and in vivo, that QKI-6 functions in the same manner as GLD-1 and can specifically bind to TGEs to repress translation of reporter constructs containing TGEs. In addition, expression of QKI-6 in C. elegans wild-type hermaphrodites or in hermaphrodites that are partially masculinized by a loss-of-function mutation in the sex-determining gene tra-3 results in masculinization of somatic tissues, consistent with QKI-6 repressing the activity of tra-2. These results strongly suggest that QKI-6 may control gene activity by operating through TGEs to regulate translation. In addition, our data support the hypothesis that other STAR family members may also be TGE-dependent translational regulators.quaking ͉ myelination ͉ mouse ͉ translation control ͉ tra-2 and GLI element (TGE)

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 ...

Adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine in double-stranded regions of RNA. ADAR activity is in the nucleus in Xenopus laevis stage VI oocytes, and released into the cytoplasm at oocyte maturation. We previously demonstrated that a cytoplasmic double-stranded RNA (dsRNA) binding factor(s), cytodsRBP, protects microinjected dsRNA from the ADAR released at maturation. Here we describe experiments to determine whether an endogenous dsRNA, the duplex formed between sense and antisense transcripts of basic fibroblast growth factor (bFGF), is protected in a similar manner. Consistent with the presence of cyto-dsRBP, we observed that the majority of bFGF RNA was not deaminated, before or after maturation. However, a minor fraction of the bFGF RNA was deaminated whether the RNA was isolated from stage VI oocytes or matured oocytes. Since ADAR activity is in the nucleus in stage VI oocytes, our results suggest that a fraction of the bFGF RNAs are hybridized in the nucleus and are ADAR substrates. Adenosine deaminations result in A-to-G changes in cDNAs, so we quantified the fraction of modified molecules using restriction-enzyme assays of RT-PCR products. Caveats due to recombination during RT-PCR are discussed.

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.