An RNAi-based suppressor screen identifies interactors of the Myt1 ortholog of Caenorhabditis elegans

Abstract

Oocyte maturation in all species is controlled by a protein complex termed the maturation promoting factor (MPF). MPF comprises a cyclin-dependent kinase (CDK) and its partner cyclin, and it is regulated by dueling regulatory phosphorylation events on the CDK. In Caenorhabditis elegans, the Wee1/Myt1 ortholog WEE-1.3 provides the inhibitory phosphorylations on CDK-1 that keep MPF inactive and halt meiosis. Prior work has shown that depletion of WEE-1.3 in C. elegans results in precocious oocyte maturation in vivo and a highly penetrant infertility phenotype. This study sought to further define the precocious maturation phenotype and to identify novel interactors with WEE-1.3. We found that WEE-1.3 is expressed throughout the germline and in developing embryos in a perinuclear pattern, and demonstrated that oocytes in WEE-1.3-depleted germlines have begun to transcribe embryonic genes and exhibit inappropriate expression of proteins normally restricted to fertilized eggs. In addition, we performed an RNAi suppressor screen of the infertile phenotype to identify novel factors that, when co-depleted with WEE-1.3, restore fertility to these animals. We screened ∼1900 essential genes by RNAi feeding and identified 44 (∼2% of the tested genes) that are suppressors of the WEE-1.3 depletion phenotype. The suppressors include many previously unidentified players in the meiotic cell cycle and represent a pool of potential WEE-1.3 interacting proteins that function during C. elegans oocyte maturation and zygotic development.

Keywords: EGA; WEE-1.3; fertility; oocyte maturation; suppressor.

Figure 1

WEE-1.3 is expressed throughout the adult soma, germline, and embryos. (A–G) Fluorescence micrographs of live animals expressing integrated transgene avIs147 [unc-119(+) + pAA34(wee-1.3 prom::WEE-1.3::GFP::wee-1.3 3′UTR)] or an extra chromosomal array of avEx148 [unc-119(+) + pAA10(wee-1.3 prom::GFP::WEE-1.3 + wee-1.3 3′UTR)]. (A) Adult hermaphrodite exhibits perinuclear WEE-1.3::GFP expression in the germline from the distal tip (asterisk) to the proximal oocytes (arrowhead) and in developing embryos (arrows) [avIs147 (pAA34)]. Spermatheca (SPTH) is indicated by the bracketed region. Note the distal tips from both gonad arms are shown in this image. (B) Punctate expression of WEE-1.3::GFP on the surface of oocytes in the proximal germline of a rescued wee-1.3 deletion line [wee-1.3(ok729); avIs147 (pAA34)]. (C) Sperm expression (arrow) of GFP::WEE-1.3 in the spermatheca of an avEx148 animal (pAA10). (D) Perinuclear and punctate expression of WEE-1.3::GFP throughout developing embryos of a rescued wee-1.3 deletion line [wee-1.3(ok729); avIs147 (pAA34)]. (E) Somatic head nuclei expressing WEE-1.3::GFP in a rescued wee-1.3 deletion line [wee-1.3(ok729); avIs147 (pAA34)]. (F) Somatic tail nuclei expressing WEE-1.3::GFP in a rescued wee-1.3 deletion line [wee-1.3(ok729); avIs147 (pAA34)]. (G) Larval somatic expression of WEE-1.3::GFP in a rescued wee-1.3 deletion line [wee-1.3(ok729); avIs147 (pAA34)]. (H–K) Z-stack projections of confocal images of gonads dissected from wild-type mothers, fixed, and co-stained with antibodies against WEE-1.3 (H, red in K), phospho-histone H3 (Ser10) (I, green in K), and DNA (J, blue in K). The arrow in (H) indicates WEE-1.3 expression that coats the diakinetic chromosomes in mature oocytes. Scale bars are approximately 20 μm.

Figure 2

Precocious oocyte-to-embryo transition upon WEE-1.3 RNAi depletion. (A–N) Single confocal images of live wild-type (A, C, E, G, I, K, and M) or wee-1.3(RNAi) (B, D, F, H, J, L, and N) hermaphrodites expressing GFP::MBK-2 (A, B), GFP::EGG-3 (C, D), CAV-1::GFP (E, F), GFP::CHS-1 (G, H), PGL-1::GFP (I, J), OMA-1::GFP (K, L), or CBD-1::mCherry (M, N). Brackets in (K and L) denote OMA::GFP localizing to discrete punctae. Arrows in (L) denote oocytes undergoing premature degradation of OMA-1::GFP. (O and P) Z-stack projections of live images of FIB-1::GFP; Histone H2B::mCherry hermaphrodites (FIB-1 in green and histones in red). Asterisk denotes the position of the spermatheca with oocytes to the left of the spermatheca and embryos to the right. Bracket in (O and P) denote mature oocytes. Scale bar in (A) applies to (A–J) and (M–P), and is approximately 20 μm. Scale bar in (K) applies to (K and L) and is approximately 20 μm. More than 20 animals were observed and imaged for each condition, and a representative image is shown.

Figure 3

Depletion of WEE-1.3 results in precocious onset of embryonic gene activation. The expression of the three mRNAs, vet-1, vet-4, and vet-6, is normally restricted to the early embryo; however, in gonads subjected to wee-1.3(RNAi) they are expressed precociously in the gonad. The graph shows the relative amounts of the indicated mRNAs, determined by quantitative RT-PCR, found in the gonads of adults subjected to either control or wee-1.3 RNAi. Relative abundance for each target gene on wee-1.3 RNAi treatment was calculated using the comparative Ct (ΔΔCt) method in which values were first normalized to act-1 mRNA levels and then normalized to the calibrator sample (control RNAi gonads). Each bar represents the mean of three independent biological replicates, and the error bars represent SEM. Statistics were performed using a Student’s t-test. *P-value <0.05; **P-value <0.01; and ***P-value ≤0.0001.

Figure 4

Suppression screen of the WEE-1.3 RNAi infertility results in the identification of 44 candidate suppressors. (A) Pie chart summarizing the suppression screen data in which 1874 RNAi clones were screened to identify suppressors of the WEE-1.3 infertility phenotype. 92.0% (n = 1723) failed to suppress, 5.0% (n = 94) weakly suppressed, 2.5% (n = 47) moderately suppressed, and 0.5% (n = 10) strongly suppressed the WEE-1.3 RNAi depletion infertility phenotype. (B) Quantification of the degree of suppression of 51 of the moderate and strong candidates identified in Figure 4A. Average brood size per individual mother treated with wee-1.3(RNAi), control(RNAi), or co-depleted of wee-1.3 and candidate suppressor RNAi. Class 1 suppressors are denoted by a red bar and class 2 suppressors are denoted by a gray bar. Each bar represents the average of at least three independent experiments, and the errors bars represent SEM. The total n per each condition was at least 24 and at most 100 animals. Statistics were performed using a Student’s t-test; all values are P-value <0.005 when compared with the brood exhibited by wee-1.3/control RNAi-treated animals, except *P-value <0.05; n.s. indicates not significant. **Indicates a global RNAi suppressor.

Figure 5

Co-depletion of WEE-1.3 and CDK-7 suppresses the infertility phenotype of WEE-1.3 depletion. (A) Schematic on how CDK-7 (CAK or Cdk-activating kinase) and WEE-1.3 (inhibitory kinase) act on maturation promoting factor (MPF) to regulate the activity of MPF. (B) Average brood size per mother treated with control RNAi, wee-1.3 RNAi, cdk-7 RNAi, or co-depleted of wee-1.3 and cdk-7 RNAi or wee-1.3 and control RNAi. Each bar represents the average of four independent experiments with three animals per experiment (n = 12), and the error bars represent SEM. (C–H) Single-plane confocal images of gonads dissected from mothers treated with the indicated RNAi, fixed, and co-stained with antibodies against phosphohistone H3 (Ser10) (pH3, green), nucleolus (Nop1p, red), and DNA (DAPI, blue). RNAi treatment is as follows: (C) control; (D) WEE-1.3–depleted; (E) CDK-7–depleted; (F) co-depletion of WEE-1.3 and control; (G) co-depletion of CDK-7 and control; and (H) co-depletion of WEE-1.3 and CDK-7. Gonads are oriented with the proximal region to the right in this figure. Scale bar is approximately 20 μm. Individual panels of Nop1p and pH3 antibody staining can be found in Figure S3.