Fig. 6. Osk prevents nos mRNA deadenylation by preventing the binding of Smg to nos mRNA. (A) PAT assays of nos mRNA showing that poly(A) tails of bulk nos mRNA are not affected by the lack of Osk. Df is Df(3R)Exel6198. osk females were crossed with wild-type males and twin or osk twin females were crossed with identical males. sop mRNA was used as a control in A and B. (B) PAT assays showing that nos mRNA has longer poly(A) tails and is stabilized in embryos where Osk protein is overexpressed ubiquitously (UASp-osk-K10/+; nos-Gal4/+). Females were crossed with wild-type males. (C) Smg immunoprecipitations in 0-3 hour embryo extracts, either from wild-type embryos, or from embryos in which osk is overexpressed (UASp-osk-K10/+; nos-Gal4/+). Ribonucleoprotein complexes were precipitated with anti-Smg (Smg IP) or rabbit serum (mock IP) in the presence of RNAsin. Bound proteins were revealed by western blots with anti-Smg and anti-CAF1 (top and middle panels). Ubiquitous expression of osk does not affect CAF1 co-immunoprecipitation with Smg. Bound RNAs were analysed by RT-PCR to visualize and quantify nos mRNA versus sop or rp49 mRNAs used as controls. nos and the control mRNA (sop or rp49) were analysed in the same PCR reaction. An example of nos mRNA enrichment in Smg IP is shown (wild type); this enrichment is lost in Smg IP from embryos where osk is overexpressed (UASp-osk-K10/+; nos-Gal4/+) (bottom panel). Protein and RNA extracts before immunoprecipitation were also loaded (Extract). (D) Quantification of nos mRNA enrichment in Smg IP. PCR were performed on several dilutions of the RT reactions. The levels of sop or rp49 control mRNAs were set at 1 and the levels of nos mRNA were calculated (nos mRNA/sop or rp49 mRNA). The fold enrichment of nos mRNA in Smg IP compared to mock IP is indicated (ratio of nos mRNA level in Smg IP to that in mock IP). Quantifications were from four RT-PCR and three QRT-PCR in one set of immunoprecipitations. Similar results were obtained from an independent set of immunoprecipitations (mean of six RT-PCR: 2.9 in wild-type embryos, versus 1.3 in embryos overexpressing osk).