Phosphorylation of Cyclin A. (A) Protein extracts from wild-type ovaries, embryos or embryos produced by png¹⁰⁵⁸ mutant females were incubated in λ phosphatase buffer, with or without λ phosphatase and immunoblotted for Cyclin A. In all extracts treatment with λ phosphatase collapses the forms of Cyclin A to the fastest mobility form, indicating the 3 slower mobility forms are due to phosphorylation. (B) In vitro phosphorylation assay for Cyclin A. Full length and truncations of HA-Cyclin A were translated in vitro, labeled with ³⁵S methionine (lane 1 for each construct). The translated protein was incubated with embryonic extracts and the mobility forms tested (lane 2). These mobility shifts were shown to be the consequence of phosphorylation by incubation with λ phosphatase (lane 4) compared with phosphatase buffer alone (lane 3). The extracts produce the same phosphorylation shifts observed in vivo during oogenesis and embryogenesis. Lanes 5 and 6 demonstrate that png mutant extracts are capable of promoting phosporylation. (C) Diagram of the Cyclin A deletions assayed for phosphorylation (26). The C terminus of the protein is required for phosphorylation, amino acids 1–170 are required for 2 of the phosphorylations, but amino acids 1–80 are dispensable for phosphorylation. The autophosphorylation sites for cyclin A are T145, S154, and S180, shown by red X's (25).

PNG overcomes repression of Cyclin A translation. (A) Premature activation of PNG activity by induction of GNU (from a nanos GAL4 driver and a UASp gnu-GFP) results in increased levels of Cyclin A in ovaries enriched for late stage egg chambers. Strikingly, the unphosphorylated form of Cyclin A appears (arrows), whereas normally it is not seen until after the completion of meiosis (as in extracts from unfertilized eggs). The immunoblots were probed also with antibodies against Cyclin B to confirm that it too is increased by induction of GNU, and against Tubulin as a loading control. (B) PAT assay on cyclin A mRNA isolated from wild-type ovaries or embryos laid by wild type, png, plu or gnu mutant females. The cyclin A poly(A) tail is lengthened at egg activation (top arrow, compare wild type embryo to ovary, bottom arrow). The poly(A) tail does not fully lengthen in the absence of PNG kinase activity (middle arrow, png, plu and gnu mutants). (C) PNG is needed to overcome inhibition of Cyclin A translation conferred by the 3′UTR. mRNAs were transcribed in vitro from the luciferase reporter gene alone or flanked by the α-tubulin 3′UTR, the 5′ and 3′ UTRs of cyclin B, or the 3′UTR of cyclin A. These were injected into wild-type or png¹⁰⁵⁸ mutant embryos, and the level of luciferase activity assayed. The data are presented as a ratio of luciferase activity obtained from png mutant embryos relative to wild type. (D) Double mutants between png and the translational repressor pum show an increase in Cyclin A protein levels compared with png mutants and the reappearance of the unphosphorylated form. Protein extracts were prepared from embryos laid by mothers of the indicated genotype and immunoblots bound to antibodies against Cyclin A, Cyclin B, or Tubulin. Only 2 forms of Cyclin A are distinguishable under the electrophoresis conditions used in this experiment. (E) Cyclin A protein levels are decreased in png mutant eggs and in stage 14 oocytes, suggesting the PNG kinase complex functions before egg activation to promote Cyclin A levels.

Developmental regulation of Cyclin A protein. (A) Cyclin A levels and forms in staged egg chambers detected by immunoblot. Cyclin A protein is nearly undetectable during the prophase I arrest (stage 10) but abundant at maturation (stage 12). Two fast migrating forms are present premeiotically in stages 1–6, whereas from maturation through metaphase I 2 slower migrating forms appear (arrows). A shorter exposure shows the forms present in stage 12. All 4 forms of Cyclin A are present in unfertilized eggs. Tubulin serves as a loading control. (B) Immunoblot of Bruno protein. Bruno protein levels markedly decline upon oocyte maturation (stages 12–13) and are undetectable at metaphase I (stage 14). Tubulin serves as a loading control. (C) PAT assay of poly(A) tail length on cyclin A mRNA. Polyadenylation occurs at maturation (stage 12, middle arrow) and the poly(A) tail is further lengthened by metaphase I (stage 14, top arrow). The length of the poly(A) tail in each lane is demarcated by the top of the smear.

Effects of cort, mr, and png mutants on the phospho forms of Cyclin A. Protein extracts were prepared from unfertilized eggs or embryos from mothers of the indicated genotypes and immunoblots done for Cyclin A. (A) Mutation of cort leads to the accumulation specifically of the most phosphorylated form of Cyclin A. The immunoblot shown was prepared from extracts from 0–1-h embryos, but unfertilized eggs and mature oocytes give the same result. (B) png, cort epistasis test. Protein extracts prepared from unfertilized eggs from png, cort heterozygous mothers, mothers homozygous for one and heterozygous for the other, or double homozygous mutant mothers were immunoblotted for Cyclin A. In contrast to cort mutants, png mutants have low levels of Cyclin A protein, and it is the most phosphorylated form. The levels of Cyclin A protein are not restored in png, cort double mutants. The same result is obtained in embryos. (C) Epistasis tests between png and mr also do not show restoration of Cyclin A levels; png is epistatic to mr.

Model for the posttranscriptional regulation of Cyclin A in meiosis and early embryogenesis. The events affecting Cyclin A protein levels are schematized above the time line for meiosis and the restart of the cell cycle in embryogenesis. PNG begins to promote Cyclin A protein levels during oogenesis, but it is not known if Pumilio is inhibitory to Cyclin A translation at this time or only during embryogenesis. The Cort form of the APC/C targets Cyclin A for degradation during prometaphase I, but Cort is degraded after the completion of meiosis (10), so APC/C^Fzy controls Cyclin A destruction during the early embryonic divisions (40, 48, 49). PNG is needed during the early embryonic divisions for translation of Cyclin A, but this requirement is obviated if pum is mutated.