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First published online 18 October 2006
doi: 10.1242/dev.02649

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Oskar allows nanos mRNA translation in Drosophila embryos by preventing its deadenylation by Smaug/CCR4

Génétique du Développement de la Drosophile, Institut de Génétique Humaine, CNRS UPR 1142, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France.

View larger version (51K): [in a new window]   Fig. 1. Characterization of twin mutants and function in the germline. (A) Schematic representation of twin locus and mutants. Black boxes indicates exons. Intron 5 is 19.6 kb long according to genomic and EST sequences in FlyBase. The arrow indicates the transcription start site. P elements (not drawn to scale) inserted in the twin locus in the three alleles are shown. In twin12209 and twin8115, insertions are P-UAS-GFP and P-UAS, respectively. Insertion sites were verified by DNA sequencing. Coordinates of the insertion sites, according to the AE003746 sequence in NCBI, are 189597 and 173089 for twinKG877, 189529 for twin12209 and 174697 for twin8115. (B-D) Immunostaining of wild-type and twin mutant ovaries with anti-CCR4 antibody. (B) Wild-type, (C) twin12209 and (D) twin8115 stage 10 egg chambers, stained with anti-CCR4 (left) and DAPI to visualize DNA (right). (E) twin mutant females show maternal effect embryonic lethality. Females of the indicated genotypes were crossed with wild-type males and hatched and unhatched embryos were scored. A proportion of these embryos have a thin chorion. When possible, both twin homozygous females and twin alleles in combination with Df(3R)Exel6198 were analysed. twinKG877 homozygous are lethal at larval stage, due to an independent mutation on the chromosome (Temme et al., 2004). Df(3R)Exel6198 is a deficiency overlapping the twin locus, which is independent and shorter than Df(3R)crb-F89-4. The phenotypes of twin12209/Df(3R)Exel6198 and twin8115/Df(3R)Exel6198 are stronger than that of homozygous twin12209 and twin8115 homozygous females, respectively, indicating that none of these alleles is null. (F-K) Ovarian phenotypes of twin mutant females were analysed by DAPI staining. (F) Stage 3 to 10 egg chambers were scored according to their numbers of cells. Df is Df(3R)crb-F89-4. Similar results were obtained when Df(3R)Exel6198 was used (wt, wild type; other: phenotypes including no oocytes, two oocytes or one mislocalized oocyte). (G) Wild-type egg chambers. (H,I) twin12209/Df(3R)crb-F89-4. An example of 32 germline cell phenotype and of an apoptotic egg chamber are shown in H and I, respectively. (J,K) Staining with DAPI and anti-HtsRC antibody that recognizes ring canals. The oocyte is linked to four nurse cells by four ring canals in wild-type egg chambers (J, arrow). An example of twin12209/Df(3R)crb-F89-4 egg chamber, where the oocyte is linked by five ring canals (K, arrow). (L,M) Mitosis defects in embryos from twin mutant females visualized by DAPI staining. Nuclear cleavages are synchronous in wild-type syncytial blastoderm embryos (L), whereas nuclei in all the phases of the cell cycle are found in a same embryo from twinKG877/Df(3R)crb-F89-4 female (M). Bottom panels show high magnifications of images in L,M. Anterior is oriented toward the left in all panels.

View larger version (41K): [in a new window]   Fig. 2. CCR4 and Smg are required for nos mRNA deadenylation and degradation. (A) PAT assays and RT-PCR of nos mRNA showing its deadenylation and destabilization in the wild type and its stabilization in twin mutants during early embryogenesis. Females of the indicated genotypes were crossed with identical males. Note that, consistent with both maternal and zygotic contributions to mRNA destabilization in early embryos (Bashirullah et al., 1999), we found that embryonic lethality increased when twin females were crossed with males of the same genotype instead of wild-type males [e.g. 94% embryonic lethality (n=468) from a cross between twin12209 homozygous females and males]. Df is Df(3R)crb-F89-4. RNAs were from embryos spanning 1 hour intervals. The sop mRNA (bottom panels) was used as a control in A and B. (B) PAT assays of nos mRNA showing its stabilization with long poly(A) tails in smg mutants or from a nos transgene lacking the TCE. nosBN is a null mutant that does not produce nos RNA. Females of the indicated genotypes were crossed with wild-type males. RNA was prepared from the time intervals indicated. (C) In situ hybridizations revealing nos mRNA in embryos. twinKG877/Df(3R)crb-F89-4 females were crossed with identical males and smg1/Df(ScfR6) females were crossed with wild-type males. Anterior is oriented toward the left.

View larger version (37K): [in a new window]   Fig. 3. Genetic and physical interactions between Smg and the CCR4-NOT complex of deadenylation. (A) Genetic interaction between smg and twin. Females of the indicated genotypes were crossed with wild-type males, and hatched and unhatched embryos were scored. (B) PAT assays of nos and control sop mRNAs showing a wild-type pattern of nos mRNA poly(A) tails in embryos from smg1/+ or twin8115/+ females and a stabilization of nos mRNA with longer poly(A) tails in embryos from smg1/+ twin8115/+ double heterozygous females; poly(A) tails were still longer in embryos from double homozygous smg1 twin12209 mutant females (right panel). Females were crossed with identical males. (C) Co-immunoprecipitation of CAF1 protein with Smg in 0-3 hour embryo extracts. Proteins were immunoprecipitated with anti-Smg (Smg IP) or rabbit serum (mock IP) either in the presence or the absence of RNase A. Bound proteins were detected by western blots with anti-Smg or anti-CAF1. Extract before IP was also loaded.

View larger version (36K): [in a new window]   Fig. 4. Deadenylation of nos mRNA by CCR4 contributes to its translational repression. (A) Immunostaining of embryos with anti-Nos antibody during the first hours of embryogenesis. The increase in amounts of Nos protein was visible in bulk embryos from twinKG877/Df(3R)crb-F89-4 and twin12209/Df(3R)crb-F89-4 females. twin mutant females were crossed with identical males and smg1/Df(ScfR6) females were crossed with wild-type males. (B-D) Cuticle preparations of embryos showing strong head defects. (B) Wild type. (C,D) Embryos from twin12209/Df(3R)crb-F89-4 females crossed with wild-type males; (C) head replaced by a hole; (D) no head structures. Anterior is oriented toward the left.

View larger version (49K): [in a new window]   Fig. 5. Characterization of embryonic phenotypes caused by overexpression of osk with UASp-osk-K10. (A,B) Cuticle of embryos from UASp-osk-K10/+; nos-Gal4/+ females were prepared and classified from their defects (n=155). Examples of the different phenotypes are shown in B. Two different examples of strong head defects (25%) are shown: head replaced by a hole (middle top panel), no head structures (middle bottom panel). The phenotype of mirror posterior duplication (51%) is also shown (bottom panel). (C) Immunostaining of embryos from wild-type or UASp-osk-K10/+; nos-Gal4/+ females with anti-Nos antibody, showing that Nos protein accumulates in the whole embryo when osk is overexpressed ubiquitously. Anterior is oriented toward the left.

View larger version (61K): [in a new window]   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).

View larger version (93K): [in a new window]   Fig. 7. Presence of Smg and of the CCR4-NOT complex in discrete cytoplasmic foci related to P bodies. Immunostaining of wild-type syncytial blastoderm embryos (90-110 minute development) with anti-CAF1, anti-CCR4, anti-Pacman or anti-Dcp1, costained with anti-Smg and DAPI (not shown). Pacman and Smg are exclusively cytoplasmic, whereas CAF1, CCR4 and Dcp1 are present in low amounts in nuclei, in addition to their cytoplasmic distribution. All proteins show a diffuse cytoplasmic distribution and accumulation in cytoplasmic foci variable in size. Arrows indicate medium size foci where either CAF1, CCR4 or Pacman colocalize with Smg. Arrowheads indicate large size Smg foci that do not contain CAF1, CCR4 or Pacman, but do contain Dcp1.