Cell type-specific translational repression of Cyclin B during meiosis in males

Abstract

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I.

Keywords: Cyclin B; Drosophila; Meiosis; Spermatogenesis; Translational control.

Fig. 1.

Rbp4 and Fest are expressed shortly after the onset of spermatocyte development and interact physically. (A) Schematic of germ cell development in the Drosophila male. Due to space constraints, only one spermatocyte (red brackets) is shown advancing through the meiotic and post-meiotic stages. Orange, hub; blue, germline stem cell; gray, spermatogonia; dark red, germ cells undergoing premeiotic S DNA replication; turquoise, spermatocytes; yellow, meiotic divisions; green, round spermatids. (B,C) Anti-GFP immunofluorescence of testes from flies expressing (B) Rbp4-eYFP or (C) eYFP-Fest. Scale bars: 100 µm. (D,E) Anti-GFP (green) and anti-HA (red) immunofluorescence on testes from flies expressing (D) Rbp4-eYFP, Bam-HA or (E) eYFP-Fest, Bam-HA. Scale bars: 50 µm. (F) RT-PCR on fest transcript from RNA from males, females and gonadectomized males. Control: GAPDH2. (G) Anti-GFP, anti-HA western blot of anti-GFP immunoprecipitations from testes of Rbp4-HA flies and eYFP-Fest, Rbp4-HA flies. (H) Anti-HA, anti-Myc western blot of anti-HA immunoprecipitations (left panels) or anti-Myc immunoprecipitations (right panels) from S2 cells transfected with either HA-Fest, Myc-Rbp4 or both, as indicated.

Fig. 2.

Rbp4 and Fest repress CycB accumulation in immature spermatocytes. (A-C) Anti-CycB immunofluorescence on wild-type (A), rbp4 mutant (B) and fest mutant testes (C). Yellow lines mark the region containing immature spermatocytes. Scale bar: 100 µm. (D-F) In situ hybridization on wild-type (D), rbp4 mutant (E) and fest mutant (F) testes with antisense cycB probe. Scale bar: 100 µm. (G-I) Anti-PH3Thr3 immunofluorescence on wild-type (G), rbp4 mutant (H) and fest mutant (I) testes. Scale bars: 100 µm. (G′,H′) Higher magnification of boxed areas in G,H. Scale bars: 20 µm.

Fig. 3.

Fest is required for proper entry into metaphase of meiosis I. (A-G″) Live squashes of wild-type (A-C″) and fest mutant (D-G″) testes, both expressing Tubulin-GFP. (A-G) Phase images. (A′-G′) Tub-GFP. Arrows in A′,B′,C′ indicate spindle poles. (A″-G″) Hoechst (DNA). Arrowheads indicate condensed chromosomes. (A-A″) Wild-type prometaphase of meiosis I. (B-B″) Wild-type metaphase I. (C-C″) Wild-type anaphase I. (D-D″) fest mutant spermatocytes showing condensed chromosomes (D″) but no evidence of microtubule asters (D′, compare with A′). (E-E″) A rare class of fest mutant spermatocytes with chromosomes condensed and congressed to the center of the cell (E″) but lacking a metaphase spindle (E′, compare with B′). (F-F″) fest spermatocytes with dense bundles of microtubules and nuclei in the process of getting squashed (arrowheads, F′). (G-G″) The terminal phenotype of fest mutant germ cells. Microtubules are dramatically dense and disorganized, compressing the still-arrested nucleus (arrowheads, G′). Scale bar: 50 µm in A″.

Fig. 4.

Sequences in the cycB 3′ UTR are required for translational repression. (A) 3′ RACE PCR on the cycB transcript from RNA collected from wild-type testis, wild-type ovary, bam mutant testis (spermatogonia accumulate, and spermatocytes and spermatids are absent). Forward primer for 3′ RACE was nearly identical to primer #1 in C, just 4 bases longer. (B) RT-PCR from RNA collected from wild-type testis, wild-type ovary, bam mutant testis and rbp4 mutant testis, using primers #1-3, shown in C. Predicted products: 124 bp from short and long form; 227 bp from long form only. (C) Schematic of the cycB 3′ UTR as detected in ovary and spermatogonia versus in spermatocytes. NRE, Nanos response element. (D) A conserved proportion of the short cycB 3′ UTR, with mut9+5 variation created by site-directed mutagenesis (mutated nucleotides shown in lowercase). (E) Schematic of CycB-eYFP in vivo reporter. (F-J) Anti-GFP immunofluorescence on: (F) CycB-eYFP in wild-type testis, (G) CycB-eYFP in rbp4 mutant testis and (H) CycB-eYFP-mut9+5 in wild-type testis. White lines indicate early spermatocytes. Scale bar: 100 µm in H. (I,J) RT-PCR on CycB-eYFP, CycB-eYFP in rbp4 and CycB-eYFP-mut9+5 reporters. (I) Amplifying eYFP (313-bp expected product) and (control) GAPDH2 (100 bp) to assay reporter transcript levels. (J) Using an eYFP forward primer and cycB 3′ UTR reverse primers 2 and 3. Predicted products: 157 bp from short and long form; 260 bp from long form only – not detected. (K) Anti-GFP western blot of a biotin RNA pull-down from Rbp4-eYFP or Ubi-GFP testis extract. Wild-type and mutant biotin probes as indicated. (L) Quantification via ImageJ of three independent biotin RNA pull-downs from Rbp4-eYFP. The mean of the 10% input bands was set to 1; a value of 2 for the wild-type probe indicates that that probe pulled down ∼20% of the Rbp4-eYFP input. Error bars indicate s.e.m.

Fig. 5.

A 31-amino acid domain in Rbp4 is required for binding to Fest. (A) Diagram of Rbp4 protein structure and truncated Rbp4 proteins tested in S2 cells for binding to Fest. RRM, RNA recognition motif. (B) Anti-HA, anti-Myc western blot of anti-Myc immunoprecipitations (top panels) and 10% input (bottom panels) from S2 cells transfected with HA-Fest alone or HA-Fest with various Myc-Rbp4 truncations, as indicated. (C) Alignment of Rbp4 residues 245-275 with Rbp4 homologs in other species. (D,E) Anti-GFP immunostaining of Rbp4-eYFP/+ testis (D) and Rbp4-eYFP/+; fest testis (E). Scale bar: 100 µm in E for D and E. (F) Biotin RNA pull-down from Rbp4-eYFP/+, fest testis, using the wild-type 130 nt cycB 3′ UTR probe.