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doi: 10.1242/10.1242/dev.00309

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Orb and a long poly(A) tail are required for efficient oskar translation at the posterior pole of the Drosophila oocyte

Stefania Castagnetti^* and Anne Ephrussi

Developmental Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
^* Present address: Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

View larger version (31K): [in a new window]   Fig. 1. osk mRNA has a long poly(A) tail in vivo. (A) osk mRNA poly(A) tail measurement using the PAT assay previously described by Sallés et al. (Sallés et al., 1994) and Chang et al. (Chang et al., 1999). The longest detected osk poly(A) tail is about 200 A long, both in early (up to stage 5) and late (5-14) stages of oogenesis. The length of the poly(A) tail is equal to the difference between the top of the smear and the fragment of osk mRNA amplified, both indicated by arrows. (B) Measurement of the osk poly(A) tail length using an RNAseH based assay. Total ovarian RNA was hybridized to a DNA oligonucleotide complementary to the 3'-most region of the osk 3'UTR, in the presence (+) or in the absence (-) of excess oligo dT16. The maximum length present in the osk mRNA population corresponds to the difference between the top of the smear in the `-' lane and the baseline given by the `+' lane. In wild-type ovaries, the osk poly(A) tail reaches a length of 200 A, whereas in orbmel ovaries the osk poly(A) tail is shortened to about 130-150 A. Quantitation using the NIH image program shows that in wild-type ovaries 36% of osk mRNA has a tail length of 150-200 A and that this population is reduced to 4.5% in orbmel homozygous ovaries. The same amount of total RNA was processed in each sample. The result obtained for the wild-type RNA was confirmed using a second osk oligo. (C) Translation efficiency of chimeric osk-lacZ mRNAs bearing poly(A) tails of different lengths in embryo extract. Efficient translation activation was observed when a poly(A) tail longer than 200 A was added to the transcript. Tails of 36, 53, 73 and 150A in length did not activate translation. The difference in translation between A0, A36, A53, A73 and A150 can be explained by a comparable increase in stability of the transcripts. The difference in half-life between the A0 and An transcript does not explain the difference in their translation efficiency. Previous reports had suggested that osk translation was poly(A) independent (Castagnetti et al., 2000; Lie and Macdonald, 1999). In both cases the poly(A) tails used in the assay were shorter than 200 A and therefore not competent to activate translation according to our present analysis.

View larger version (27K): [in a new window]   Fig. 2. osk mRNA localization is only mildly affected in orbmel mutants. Whole-mount in situ hybridization (upper panel) was performed on ovaries from wild-type and orbmel homozygous flies, using a DIG-labeled osk probe. The result obtained by RNA in situ hybridization was confirmed by immunostaining with -Stau antibody (lower panel).

View larger version (35K): [in a new window]   Fig. 3. Osk accumulation in orbmel homozygous egg chambers. (A) Western blot analysis was performed on total ovarian protein extracts of similarly aged wild-type and orbmel flies. The reduction in Osk phosphorylation is presumably a consequence of the reduction in Osk accumulation, as Osk protein has been shown to be required for its own phosphorylation and stabilization (Markussen et al., 1997; Riechmann et al., 2002). (B) Whole-mount antibody staining of orbmel egg chambers with Stau, and Stau and Osk antibodies.

View larger version (13K): [in a new window]   Fig. 4. A long poly(A) tail is not sufficient to overcome BRE-mediated repression. Translation efficiency of chimeric osk-lacZ mRNAs bearing either no poly(A) tail or a tail of >200A in ovary extract. The transcripts used in these experiments bear the 5' region of osk mRNA up to the second AUG codon, and either the wild-type osk 3'UTR (WT) or an osk 3'UTR from which the BREs were deleted (ABC). The data shown are representative of three independent experiments. The values were normalized for those of a luciferase RNA co-translated as an internal control and the experimental error varies between 3% and 10%.

View larger version (48K): [in a new window]   Fig. 5. BicC, Orb and Bru interact in vitro. (A) Immunoprecipitation experiments were performed on total ovarian extract using -Orb (lane 1), -BicC (lane 2) or -Hrp48 (lane 3) antibodies, or beads alone (lane 4). After precipitation, proteins were detected using -Orb antibodies. Equal amounts of extract were used in each sample. No Orb protein is retained on the beads alone (lane 4) or with -Hrp48 (lane 3) antibody. Orb is readily precipitated by -Orb (lane 1) and -BicC (lane 2) antibodies. (B) Bru protein is pulled down from total ovarian extract by the -Orb antibody. The same blot was probed with -Orb (1) and -Bru antibodies (2).

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