A developmentally regulated translational control pathway establishes the meiotic chromosome segregation pattern

Abstract

Production of haploid gametes from diploid progenitor cells is mediated by a specialized cell division, meiosis, where two divisions, meiosis I and II, follow a single S phase. Errors in progression from meiosis I to meiosis II lead to aneuploid and polyploid gametes, but the regulatory mechanisms controlling this transition are poorly understood. Here, we demonstrate that the conserved kinase Ime2 regulates the timing and order of the meiotic divisions by controlling translation. Ime2 coordinates translational activation of a cluster of genes at the meiosis I-meiosis II transition, including the critical determinant of the meiotic chromosome segregation pattern CLB3. We further show that Ime2 mediates translational control through the meiosis-specific RNA-binding protein Rim4. Rim4 inhibits translation of CLB3 during meiosis I by interacting with the 5' untranslated region (UTR) of CLB3. At the onset of meiosis II, Ime2 kinase activity rises and triggers a decrease in Rim4 protein levels, thereby alleviating translational repression. Our results elucidate a novel developmentally regulated translational control pathway that establishes the meiotic chromosome segregation pattern.

Keywords: RNA-binding protein; cyclins; gametogenesis; kinase; meiosis; translational control; untranslated region.

Figure 1.

5′ UTR-mediated translational control of CLB3 is compromised when Ime2 is hyperactivated. (A) CLB3-3HA (A15055) and CLB3-3HA IME2st (A28184) strains carrying the GAL4.ER and pGAL-NDT80 fusions were induced to sporulate at 30°C. Six hours after transfer into sporulation-inducing conditions, when cells were arrested in the prophase I block, cells were released by the addition of 1 μM β-estradiol. Clb3-3HA and Pgk1 (loading control) protein and CLB3 RNA and rRNA (loading control) levels were determined at the indicated times. Quantification of Clb3 (Clb3/Pgk1) is shown in red, and quantification of CLB3 (CLB3/rRNA) is shown in blue. The percentage of metaphase I (MI), anaphase I (AI), metaphase II (MII), and anaphase II (AII) cells was also determined by tubulin immunofluorescence. (B) pGAL-5′ UTR_CLB3CLB3-3HA (A28887) and pGAL-5′ UTR_CLB3CLB3-3HA IME2st (A28164) strains carrying the GAL4.ER and pGAL-NDT80 fusions were analyzed as in A. (C) CLB3-3HA (A15055), 5′ UTRΔ-CLB3-3HA (A29363), and 5′ UTRΔ-CLB3-3HA IME2st (A28806) strains carrying the GAL4.ER and pGAL-NDT80 fusions were analyzed as in A.

Figure 2.

Ime2 mediates the translational control of a gene cluster during meiosis I. Cells carrying the GAL4.ER and pGAL-NDT80 alleles were induced to sporulate as described in Figure 1A. Clb3-3HA protein (red) and CLB3 RNA (blue) levels and meiotic progression were determined at the indicated times. In addition, SPO20 (A27537 and A29215) (A), GIP1 (A28121 and A29128) (B), SPS1 (A28127 and A28829) (C), and SPO21 (A30047 and A30572) (D) protein (red) and RNA (blue) levels were analyzed.

Figure 3.

Translational repression is acutely responsive to Ime2 levels in prophase I-arrested cells. IME2 (A29221) (A) and IME2st (A28789) (B) strains carrying the GAL4.ER, ndt80Δ, and pGAL-5′ UTR_CLB3CLB3-3HA alleles were induced to sporulate at 30°C and treated with 1 μM β-estradiol after 6 h to induce CLB3 expression. Clb3-3HA protein (red) and CLB3 RNA (blue) levels as well as the percentage of cells harboring two spindle pole bodies (2 SPBs) were analyzed. The images show spindle (red channel) and nuclear (blue channel) morphology of representative cells that either were arrested in prophase (left) or had formed a spindle (right).

Figure 4.

A novel synchronization method to analyze CLB3 translational control. (A) Wild-type (A4962) and pCUP1-IME1 pCUP1-IME4 (A33366) strains were induced to sporulate at 30°C. After 2 h, 25 μM CuSO₄ was added, and DNA content was determined at the indicated times. (B–D) Wild-type (A33671) and pCUP1-IME1 pCUP1-IME4 (A33432) strains carrying the CLB3-3HA and IME2-3V5 alleles were induced to sporulate as described in A. At the indicated times, DNA content (B), spindle morphology (C), and Ime2 (orange) and Clb3 (red) protein and CLB3 RNA (blue) levels were analyzed.

Figure 5.

Rim4 regulates translation of the IME2-regulated gene cluster. (A) Wild-type (A15055), rim4F139L-3V5 (A31420), and rim4F349L-3V5 (A31421) strains carrying the GAL4.ER, pGAL-NDT80, and CLB3-3HA alleles were sporulated at 30°C. After 6 h, cells were released from the prophase I arrest, and Clb3 protein (red) and RNA (blue) levels as well as spindle morphology were determined at the indicated times. (B) Wild-type (A29221), RIM4-3V5 (A32978), rim4F139L-3V5 (A33322), and rim4F349L-3V5 (A33429) strains carrying the GAL4.ER, ndt80Δ, ZIP1-GFP, and pGAL-5′ UTR_CLB3CLB3-3HA alleles were grown as in A to analyze Rim4-3V5 and Clb3-3HA protein (red) and CLB3 RNA (blue) levels. Spindle morphology as well as the presence or absence of Zip1 were determined (see Supplemental Fig. S6D for classification criteria). (C) Rim4 binds the CLB3 5′ UTR. Extracts from GAL4.ER, pGAL-NDT80, RIM4-3V5, and CLB3-3HA cells (A30868) arrested in prophase I were incubated with in vitro transcribed, 3′ biotinylated RNAs conjugated to streptavidin Dynabeads. Beads were recovered, and RNase elution was performed to release bound factors (Michlewski and Caceres 2010). Shown is the amount of Rim4 purified using each RNA bait. Quantifications are shown as fold enrichment over the no-RNA control. (D) Rim4 binds the IME2-regulated gene cluster mRNAs. RIM4-3V5 (A30868) and rim4-F139L-3V5 (A31420) strains carrying the GAL4.ER, pGAL-NDT80, and CLB3-3HA alleles were sporulated at 30°C. After 6 h, cells were released from the prophase I arrest, and 800 mL of cell pellets was collected at meiosis I (7.5 h). Rim4 was purified from extracts using anti-V5 agarose and eluted with 2.5 mg/mL V5 peptide. RNA was extracted from the eluate and converted to cDNA and then to cRNA. Immunoprecipitated RNA from both RIM4-3V5 and rim4-F139L-3V5 was labeled with Cy3, and reference RNA was labeled with Cy5. Total RNA extracted from RIM4-3V5 cells in meiosis I was used as the reference RNA. Cy3- and Cy5-labeled RNA was hybridized to an expression array. Shown is a mathematical comparison of RIM4-3V5 versus rim4-F139L-3V5 normalized using the common reference RNA. The Y-axis shows the log₂ enrichment of each gene (RIM4-3V5/rim4-F139L-3V5), and expression level is shown on the X-axis. CLB3 is highlighted in dark red; GIP1, SPS1, YSP2, YFL012W, and SPO20 are highlighted in red; translationally repressed genes that cocluster with AMA1 are highlighted in yellow; and a negative control pool of nontranslationally repressed, highly expressed genes is highlighted in green.

Figure 6.

Inhibition of Ime2 causes stabilized Rim4 and translational inhibition of CLB3. RIM4-3V5 IME2 (A30868 ± 1-NA-PP1), RIM4-3V5 ime2-as1 (A33601 ± 1-NA-PP1), rim4F139L-3V5 IME2 (A31420 ± 1-NA-PP1), and rim4F349L-3V5 ime2-as1 (A33748 ± 1-NA-PP1) strains carrying the GAL4.ER, pGAL-NDT80, and CLB3-3HA alleles were grown as described in Figure 1A. At the time of release from the prophase I block, cultures were split and received either 20 μM 1-NA-PP1 (Ime2-as1 inhibitor) or DMSO (control). (A) Rim4 levels in metaphase I (MI), anaphase I (AI), metaphase II (MII), and anaphase II (AII). Rim4 (red), tubulin (green), and DNA (blue) are shown. Exposures are identical for each image. (B) Quantification of Rim4 levels in RIM4-3V5 IME2 ([orange] − inhibitor; [green] + inhibitor) and RIM4-3V5 ime2as1 ([blue] − inhibitor; [red] + inhibitor) cells. Shown is the percentage of Rim4-positive cells (10-fold intensity greater than a no-tag control) by meiotic cell cycle stage. Meiotic prophase (P), metaphase I (MI), anaphase I (AI), metaphase II (MII), anaphase II (AII), and post-meiotic (PM) are shown. (C,D) Rim4-3V5 and Clb3-3HA protein (red) and CLB3 RNA (blue) levels as well as meiotic progression were analyzed.

Figure 7.

Ime2 kinase activity is up-regulated in meiosis II and affects Rim4 abundance. (A–C) Expression of hyperactive Ime2 affects Rim4 mobility. IME2 (A30868) and IME2st (A33027) strains carrying the GAL4.ER, pGAL-NDT80, CLB3-3HA, and RIM4-3V5 alleles were induced to sporulate as described in Figure 1A. Rim4-3V5 and Clb3-3HA protein and CLB3 RNA levels (A), meiotic progression (B), and Rim4 abundance in individual cells (C) were determined at the indicated times. (D) Shown is intensity of Rim4 signal by immunofluorescence (dashed lines) and the intensity of Clb3 by immunoblot quantification (solid lines) over the meiotic time course in the IME2 (orange) and IME2st (purple) backgrounds. (E,F) IME2-3V5 (A27742) (E) and IME2st-3V5 (A28342) (F) strains carrying the GAL4.ER, pGAL-NDT80, and CLB3-3HA alleles were induced to sporulate as described in Figure 1A. Ime2 kinase activity (orange), Ime2 immunoprecipitation, Clb3-3HA protein (red), and CLB3 RNA (blue) levels and meiotic progression were determined at the indicated times.