Multiple Mechanisms Inactivate the LIN-41 RNA-Binding Protein To Ensure a Robust Oocyte-to-Embryo Transition in Caenorhabditis elegans

Abstract

In the nematode Caenorhabditis elegans, the conserved LIN-41 RNA-binding protein is a translational repressor that coordinately controls oocyte growth and meiotic maturation. LIN-41 exerts these effects, at least in part, by preventing the premature activation of the cyclin-dependent kinase CDK-1 Here we investigate the mechanism by which LIN-41 is rapidly eliminated upon the onset of meiotic maturation. Elimination of LIN-41 requires the activities of CDK-1 and multiple SCF (Skp1, Cul1, and F-box protein)-type E3 ubiquitin ligase subunits, including the conserved substrate adaptor protein SEL-10/Fbw7/Cdc4, suggesting that LIN-41 is a target of ubiquitin-mediated protein degradation. Within the LIN-41 protein, two nonoverlapping regions, Deg-A and Deg-B, are individually necessary for LIN-41 degradation; both contain several potential phosphodegron sequences, and at least one of these sequences is required for LIN-41 degradation. Finally, Deg-A and Deg-B are sufficient, in combination, to mediate SEL-10-dependent degradation when transplanted into a different oocyte protein. Although LIN-41 is a potent inhibitor of protein translation and M phase entry, the failure to eliminate LIN-41 from early embryos does not result in the continued translational repression of LIN-41 oocyte messenger RNA targets. Based on these observations, we propose a model for the elimination of LIN-41 by the SEL-10 E3 ubiquitin ligase and suggest that LIN-41 is inactivated before it is degraded. Furthermore, we provide evidence that another RNA-binding protein, the GLD-1 tumor suppressor, is regulated similarly. Redundant mechanisms to extinguish translational repression by RNA-binding proteins may both control and provide robustness to irreversible developmental transitions, including meiotic maturation and the oocyte-to-embryo transition.

Keywords: RNA-binding proteins; oocyte meiotic maturation; oocyte-to-embryo transition; translational regulation; ubiquitin-mediated protein degradation.

Figure 1

GFP::LIN-41 is eliminated during the first meiotic division. (A and B) Composite GFP (A) and DIC (B) images of a lin-41(tn1541[gfp::tev::s-tag::lin-41]) adult hermaphrodite. GFP::LIN-41 is apparent in the middle and proximal regions of the germline (solid outline, A), with reduced levels in the −1 oocyte immediately adjacent to the spermatheca (sp). The positions of some embryos (dashed outlines, A) and oocytes are indicated relative to the spermatheca in B; a fertilized embryo in the spermatheca would be at the zero position. These labels and naming conventions are used throughout. 100 ms GFP exposures; Bar, 50 μm. (C–G) Time-lapse images of GFP::LIN-41 (white) and mCHERRY::HISTONE-labeled chromosomes (red) were acquired in a living lin-41(tn1541); itIs37[pie-1p::mCherry:::H2B::pie-1 3′UTR, unc-119(+)] adult hermaphrodite by confocal microscopy. Images are shown for select time points (t) prior to meiotic maturation (C, t= −4.5 min), at ovulation (D, t = 0 min), and during the first meiotic division (E, t= +4 min; F, t = +11.8 min; G, t= +16.9 min) as an individual oocyte (C, solid outline) progresses from the −1 to the +1 position and through the OET (D–G, dashed outlines). Bar, 50 μm. Movie S1, worm 1, shows the complete time-lapse series from which the still images were taken. (H) Five oocytes were imaged as they progressed from the −1 position through meiotic divisions; the relative amount of background-corrected GFP::LIN-41 with respect to distal oocytes is shown on the graph at each time point. Three of the oocytes were also imaged at earlier stages as they moved from a more distal location [−2 oocyte (red) or −3 oocyte (green) position] into the −1 oocyte position (blue), as indicated. Timing on the x-axis is relative to ovulation (t = 0). Bars indicate the SD for different meiotic events. Ana, anaphase; Met, metaphase; NEBD, nuclear envelope breakdown.

Figure 2

LIN-41 Deg domains are required for the elimination of GFP::LIN-41 upon the onset of meiotic maturation. (A) The exon–intron structure and deletion analysis of lin-41(tn1541). Colored boxes indicate exonic regions that encode GFP (green) or LIN-41 protein domains (see B). Deletions made in the context of lin-41(tn1541) are drawn as lines, labeled with a deletion-specific allele name, beneath the LIN-41-encoding exons and introns (exons labeled 1–15). GFP::LIN-41 can be detected in the germline of most deletion mutants (solid lines), with one exception (tn1628, dotted line). Deletions in red prevent the elimination of GFP::LIN-41 from early embryos. The vertical dashed lines delimit the beginning of Deg-A and the end of Deg-B, respectively. (B) The previously described [RING (yellow), B-box (gray), BBC (orange), Ig/filamin (purple), NHL (blue)] and newly identified [Deg (red)] protein domains of LIN-41. The vertical dashed line in B indicates the two parts of Deg-B, B1 and B2, which are individually removed in lin-41(tn1541tn1635) and lin-41(tn1541tn1622), respectively. The position of two lin-41 missense alleles (tn1767[T83A] and tn1487ts[D1125N]) generated in the endogenous lin-41 locus are indicated. (C) The amino acid sequences of Deg-A, Deg-B1, and Deg-B2. Many of the amino acids are serines and threonines (underlined) and some are potential targets of proline-directed serine/threonine [S/T] kinases (bold). The indicated alleles change an [S/T] residue to an alanine (colored and bold) in the context of lin-41(tn1541). The tn1645[T83A] mutation in Deg-A results in the persistence of GFP::LIN-41[T83A] in embryos (red), whereas the other changes do not (indicated in blue font). (D–G) GFP::LIN-41 is eliminated from the early embryos (dashed outlines) of lin-41(tn1541) (D, control) and lin-41(tn1541tn1630) (E, RING deleted) homozygous mutants but persists in the early embryos of lin-41(tn1541tn1638) (F, Deg-A deleted) and lin-41(tn1541tn1645) (G, LIN-41[T83A]) homozygous mutants. The position of the spermatheca (sp) is indicated, for reference. 100 ms GFP exposures; Bar, 20 μm. (H) The rate of ovulation is slightly reduced in mutants with a compromised LIN-41 Deg-A domain. Ovulation rate is expressed as the number of ovulations per gonad arm per hr and was measured in at least 25 day 2 adults. The three gfp-tagged alleles are in the top portion of the graph with green shading. Significance was determined using a one-way ANOVA test with Tukey’s post hoc test to compare the means; ** P < 0.01, *** P < 0.001, and **** P < 0.0001. itIs37[pie-1p::mCherry:::H2B::pie-1 3′UTR, unc-119(+)] was also present in each of the GFP::LIN-41-expressing strains; it is not expected to alter the ovulation rate.

Figure 3

LIN-41 degradation domains when implanted into mNG::OMA-2 promote its rapid elimination during meiosis. (A–C) The exon–intron structures of oma-2(cp145[mng::tev::3xflag::oma-2]), oma-2(tn1760[mng::tev::3xflag::deg-a::oma-2]), and oma-2(tn1764[mng::tev::3xflag::deg-a::deg-b::oma-2]). Boxes represent exonic regions that encode mNeonGreen (green), the tobacco etch virus cleavage site (TEV; dark gray), FLAG epitope tags (light gray), LIN-41 Deg-A and Deg-B domains (red), the likely TAF-4 interaction domain of OMA-2 (dark blue), two OMA-2 CCCH zinc fingers (white), and other OMA-2 coding sequences (cyan). The position of LIN-41 T83 within the LIN-41 Deg-A domain is indicated by an asterisk. (D–K) GFP (D–G) and DIC (H–K) images of oma-2(cp145) (D and H), oma-2(tn1760) (E and I), oma-2(tn1764) (F and J), and oma-2(tn1764) lon-3(e2175) sel-10(ar41) (G and K) one-cell embryos at pronuclear meeting (E and I), or just slightly later, as the pronuclei begin a counterclockwise rotation (D, F–G, H, J, and K) prior to nuclear envelope breakdown and the first mitotic division. Part of a −1 oocyte is visible in F and J, and is indicated for reference. 150 ms GFP exposures; Bar, 10 μm. (L–O) Time-lapse images of mNG::Deg-A,B::OMA-2 (white) and mCHERRY::HISTONE-labeled chromosomes (red) were acquired in a living oma-2(tn1764); itIs37[pie-1p::mCherry:::H2B::pie-1 3′UTR, unc-119(+)] adult hermaphrodite by confocal microscopy. Images are shown for select time points (t) at ovulation (L, t = 0 min), during the first (M, t = +5 min; N, t = +10.5 min) and second meiotic divisions (O, t = +24.5 min) as an embryo (dashed outline) progresses through both meiotic divisions. See Movie S3 for the complete time-lapse sequence. Bar, 50 μm. (P) A visual summary of the dynamic expression patterns of mNG::OMA-2 (cyan), GFP::LIN-41 (red), and mNG::Deg-A,B::OMA-2 (purple). Oocytes are to the left and embryos are to the right of the spermatheca (sp). Meiotic embryos (MI, MII) have completed their respective divisions.

Figure 4

Subunits of the SCF^SEL-10 E3 ubiquitin ligase are required for the elimination of GFP::LIN-41 from early embryos. (A–E) Composite images of GFP::LIN-41 in adult rrf-1(pk1417) lin-41(tn1541) hermaphrodites fed control RNAi bacteria (A), and adult hermaphrodites with reduced SCF^SEL-10 E3 ubiquitin ligase activity (B–E): lin-41(tn1541); skr-1(RNAi) (B), rrf-1(pk1417) lin-41(tn1541); cul-1(RNAi) (C), lin-41(tn1541); lon-3(e2175) sel-10(ar41) (D), and lin-41(tn1541); sel-10(ok1632) (E); 100 ms GFP exposures, brightened slightly (and equivalently) to better visualize embryonic GFP::LIN-41; Bar, 50 μm. (F–M) Images of two-cell embryos removed from the uterus of hermaphrodites were imaged for GFP (F–I) and DIC (J–M); the genotypes were as follows: lin-41(tn1541); lon-3(e2175) (F and J), lin-41(tn1541); lon-3(e2175) sel-10(ar41) (G and K), lin-41(tn1541) (H and L), and lin-41(tn1541); sel-10(ok1632) (I and M). Arrowheads indicate a few of the GFP::LIN-41 aggregates in the posterior blastomeres of sel-10 mutant embryos, which likely correspond to P granules; 300 ms GFP exposures; Bar, 10 μm. sp, spermatheca.

Figure 5

SEL-10 is required for the WEE-1.3–inhibited degradation of GFP::LIN-41. (A–D) Composite GFP (A and C) and DIC (B and D) images of lin-41(tn1541); lon-3(e2175); wee-1.3(RNAi) (A and B) and lin-41(tn1541); lon-3(e2175) sel-10(ar41); wee-1.3(RNAi) (C and D) animals. GFP::LIN-41 is prematurely eliminated from oocytes by wee-1.3(RNAi) (arrowhead), but persists in abnormal oocytes near the spermatheca (sp; arrow) in sel-10(ar41); wee-1.3(RNAi) animals (C and D), suggesting that SEL-10 is required for this process; 150 ms GFP exposures, brightened slightly; Bar, 50 μm. (E) A simple model for the elimination of LIN-41 (green) that incorporates the known molecular functions of WEE-1.3 kinase, cyclin-dependent kinase (CDK-1) and subunits of the SCF^SEL-10 E3 ubiquitin ligase. In brief, we hypothesize that SEL-10 (orange) may recognize phosphorylated LIN-41 (green) and trigger its ubiquitin-mediated degradation in collaboration with the other SCF E3 ubiquitin ligase subunits, SKR-1/2 (blue), and CUL-1 (blue). CUL-1 orthologs bind RING finger proteins (RBX; gray), which recruit a ubiquitin-conjugating enzyme (UBC; gray) that catalyzes the transfer of ubiquitin (yellow) to protein substrates, such as LIN-41. Subsequent recruitment of polyubiquitinated substrates to the proteasome results in degradation (not shown). This model is consistent with the epistatic relationship between wee-1.3(RNAi) and sel-10(ar41) with respect to the elimination of GFP::LIN-41, but other models are also possible.

Figure 6

Persisting LIN-41 or LIN-41[T83A] does not strongly inhibit the expression of LIN-41 targets of translational repression in young embryos. (A–J) Young embryos express similar levels of SPN-4::GFP (A, B, G, and H), GFP::MEG-1 (C, D, I, and J) and mNG::ORC-1 (arrowhead in E and F) when ectopic LIN-41[T83A] [B, D, and F; lin-41(tn1767) mutant embryos], ectopic LIN-41 [H and J; sel-10(ar41) mutant embryos] or normal (undetectable) levels of LIN-41 (A, C, E, G, and I) are present. Exposures were 100 ms for SPN-4::GFP, 200 ms for GFP::MEG-1, and 600 ms for mNG::ORC-1; Bar, 10 μm. (K and L) Quantification of the intensity of SPN-4::GFP expression in wild type, lin-41(tn1767), and sel-10(ar41) genetic backgrounds. Statistical significance was evaluated using an unpaired t-test. The plots show the mean and SD. (K) Comparison of spn-4(tn1699) and lin-41(tn1767); spn-4(tn1699) one- and two-cell embryos; no significant differences were seen (n.s.). (L) Comparison of spn-4(tn1699); lon-3(e2175) and spn-4(tn1699); lon-3(e2175) sel-10(ar41) one- and two-cell embryos. Levels appeared to be slightly lower in the sel-10(ar41) two-cell embryos (P < 0.001). Note that the slightly reduced level of SPN-4::GFP in H relative to G accurately illustrates the very modest magnitude of this difference in expression.

Figure 7

GLD-1 persists at elevated levels in the oocytes of sel-10(ar41) mutants. (A and B) Composite images of GLD-1::GFP in gld-1(q485); lon-3(e2175); ozIs2[gld-1::gfp] (A) and gld-1(q485); lon-3(e2175) sel-10(ar41); ozIs2[gld-1::gfp] (B) adult hermaphrodites. GLD-1::GFP levels remain elevated in the proximal oocytes (e.g., −4 oocytes, arrowheads) of sel-10(ar41) animals (B) relative to controls (A); 17 ms GFP exposures, brightened slightly. (C) Slow-migrating forms of GLD-1 (red arrow) are more abundant in sel-10(lf) adult hermaphrodites than in sel-10(+) controls, where the fast-migrating form of GLD-1 (black arrow) predominates. (D and E) Composite images of GLD-1::GFP in fog-3(q470); lon-3(e2175); ozIs2[gld-1::gfp] (D) and fog-3(q470); lon-3(e2175) sel-10(ar41); ozIs2[gld-1::gfp] females (E). GLD-1::GFP levels are elevated in the proximal oocytes (e.g., −4 oocytes, arrowheads) of sel-10(ar41) females (B) relative to controls (A), although this is not as dramatic as in hermaphrodites. A somewhat longer GFP exposure (35 ms, brightened slightly) was needed than in A and B, likely due to the presence of endogenous GLD-1. (F) Quantification of the intensity of GFP::MEX-3 in the proximal oocytes of lon-3(e2175); mex-3(tn1753) and lon-3(e2175) sel-10(ar41); mex-3(tn1753) hermaphrodites at 25°. No significant differences were seen (n.s.). (G and H) Composite images of lon-3(e2175); pwIs116[rme-2p::rme-2::GFP::rme-2 3′UTR] (G) and lon-3(e2175) sel-10(ar41); pwIs116 [rme-2p::rme-2::GFP::rme-2 3′UTR] (H) hermaphrodites at 22°; 300 ms GFP exposures. Neither target of GLD-1 translational repression (MEX-3, RME-2) was strongly or even marginally reduced in expression in sel-10(ar41) oocytes. (I) Quantification of the intensity of mNG::OMA-2 in the proximal oocytes of oma-2(cp145) lon-3(e2175) and oma-2(cp145) lon-3(e2175) sel-10(ar41) hermaphrodites at 20°. Differences in expression were highly significant (**** P < 0.0001), but relatively modest in magnitude. For example, we measured a 37% reduction in average fluorescence in the −2 oocytes of sel-10(ar41) animals relative to the same oocytes in control animals. Statistical tests (F and I) employed an unpaired t-test. The plots show the mean and SD. All phenotypes were analyzed on the first day of adulthood. Bar, 50 μm (A, B, D, E, G, and H). sp, spermatheca.