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 orb^mel 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 orb^mel 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 A_n 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.