Analysis of a swallow homologue from Drosophila pseudoobscura.

We analyzed a functional homologue of the swallow gene from Drosophila pseudoobscura. The swallow gene of D. melanogaster plays an essential role in localizing bicoid mRNA in oocytes, and swallow mutant embryos show anterior pattern defects that result from the lack of localization of the bicoid morphogen. The pseudoobscura homologue rescues the function of swallow mutants when introduced into the genome of D. melanogaster, and its expression is similar to that of the melanogaster gene. The predicted pseudoobscura and melanogaster proteins are 49% identical and 69% conserved. The coiled-coil domain previously identified in the melanogaster swallow protein is strongly conserved in the pseudoobscura homologue, but the weak similarity of the melanogaster swallow protein to the RNP class of RNA-binding proteins is not conserved in the pseudoobscura homologue. These and other observations suggest a structural role for swallow in localizing bicoid mRNA, perhaps as part of the egg cytoskeleton.

New phenotypes associated with the swallow gene of Drosophila: evidence for a general role in oocyte cytoskeletal organization.

During oogenesis in Drosophila, several mRNAs and proteins are localized to discrete regions of the developing oocyte, resulting in a mature oocyte with a well-defined anterior-posterior axis. The product of the swallow (sww) gene is required for the localization of two different mRNAs during oogenesis, bicoid (bcd) and Adducin-like/hu-li tai shao (hts). We initiated a detailed characterization of the phenotypes associated with each of eight sww alleles as a means of investigating the role of sww in oogenic patterning. RNA localization defects in various sww mutants were examined by radioactive in situ hybridization to paraffin sections. Using this technique, several previously unreported RNA localization defects have been observed. Although bcd RNA localization is often lost completely in sww oocytes, in a high proportion of cases, bcd RNA is localized inappropriately along the periphery of the mature oocyte. In several sww mutants, a portion of the bcd mRNA population becomes concentrated at the posterior pole of the oocyte during late oogenesis. Several sww mutations also result in oskar RNA localization defects, consistent with a global role for sww in cytoskeletal regulation or organization. A detailed temporal and spatial analysis of hts RNA localization in sww mutants and in drug-treated ovaries reveals many similarities to bcd RNA localization, and implies the two independent localization events are accomplished by the same mechanism.

Nuclear export pathways of tRNA and 40 S ribosomes include both common and specific intermediates.

Different classes of RNAs are exported from Xenopus laevis oocyte nuclei by facilitated pathways. We have performed kinetic competition analyses to investigate the relationship between the export pathways of microinjected tRNA and ribosomal subunits. Saturating concentrations of ribosomal subunits do not compete tRNA export. Thus, the saturable factor in the ribosomal subunit export pathway is not limiting for tRNA export. The co-microinjection of ribosomal subunits did, however, stimulate the rate of tRNA export. Co-injected mRNA also stimulated tRNA export. tRNA export itself displays positive cooperative export kinetics that are abrogated by saturating concentrations of rRNA. These results are consistent with the existence of common high affinity RNA-binding sites that can be titrated with tRNA, rRNA or ribosomal subunits, and mRNA. Furthermore, high concentrations of tRNA are also shown to have moderate inhibitory effects on 40 S subunit export, indicating a lower affinity common intermediate also shared by mRNA.

Microtubules are a general component of mRNA localization systems in Drosophila oocytes.

Some of the spatial cues which direct early patterning events in Drosophila embryogenesis are maternal mRNAs localized in the oocyte during oogenesis. For example, Bicaudal-D, fs (1) K10, and orb RNAs are transiently localized at the anterior oocyte margin in mid oogenesis, and oskar RNA is localized at the posterior oocyte margin beginning in mid oogenesis. Using inhibitors of cytoskeletal function, we find that microtubules, but not microfilaments, are required for localization of these mRNAs during oogenesis, results similar to those described earlier for bicoid RNA. However, the RNAs show a differential sensitivity to microtubule inhibitors. Anterior localization of Bicaudal-D, fs (1) K10, and orb RNAs is completely disrupted following even mild drug treatments. bicoid RNA localization is intermediate in its response to microtubule drugs, while oskar RNA localization is much more resistant. In addition, the localized RNAs respond differently to taxol, a microtubule stabilizing agent. The differences among these RNAs suggest that factors other than microtubules are required to maintain the positions of localized RNAs in the oocyte. Microtubules are also required for the preferential accumulation of these transcripts in the previtellogenic oocyte, consistent with the idea that these mRNAs are transported by a microtubule-dependent mechanism to the oocyte.

Localized RNAs are enriched in cytoskeletal extracts of Drosophila oocytes.

Maternal mRNAs localized within the Drosophila oocyte encode positional information which specifies the pattern of the early embryo. With the goal of identifying molecules involved in RNA localization, we have developed a subcellular fractionation procedure which enriches for localized RNAs. Most RNAs and cellular proteins are solubilized by this method and are recovered in the supernatant fraction. However, five localized RNAs we examined are recovered in the detergent-insoluble pellet, despite temporal and spatial differences in their patterns of expression. These RNAs appear to be associated with a large, detergent-insoluble component of the oocyte. This association is specific for the oocyte proper, as localized RNAs in nurse cells and early embryos do not show this behavior. The fractionation behavior of these RNAs appears directly related to their localization in the oocyte, since fractionation and localization exhibit the same cytoskeletal and genetic requirements. The cortical cytoskeleton of the oocyte is a likely candidate for the localization substratum.

Microtubules mediate the localization of bicoid RNA during Drosophila oogenesis.

We have examined cytoskeletal requirements for bicoid (bcd) RNA localization during Drosophila oogenesis. bcd is an anterior morphogen whose proper function relies on the localization of its messenger RNA to the anterior cortex of the egg. Drugs that depolymerize microtubules perturb all aspects of bcd RNA localization. During recovery from drug treatment, bcd RNA relocalizes to the oocyte cortex, suggesting that the localization machinery is a component of the cortical cytoskeleton. Taxol, a drug that stabilizes microtubules, also effectively disrupts bcd RNA localization, and the effects of taxol treatments on exuperantia and swallow mutants suggest general roles for these gene products in the multi-step bcd RNA localization process.

Sequence of swallow, a gene required for the localization of bicoid message in Drosophila eggs.

We report the sequence of the Drosophila maternal effect gene swallow, one of the genes whose product is required for the localization of bicoid message during Drosophila oogenesis. The inferred swallow protein contains a domain that is predicted to be an amphipathic alpha-helix similar to those implicated in protein:protein associations in other systems. Another part of the predicted protein appears to be a diverged RNA-binding motif. We discuss these structural features in light of the function of the swallow protein in the bicoid message localization process.