Caroline A. Spike

synMuv B proteins antagonize germline fate in the intestine and ensure C. elegans survival.

Previous studies demonstrated that a subset of synMuv B mutants ectopically misexpress germline-specific P-granule proteins in their somatic cells, suggesting a failure to properly orchestrate a soma/germline fate decision. Surprisingly, this fate confusion does not affect viability at low to ambient temperatures. Here, we show that, when grown at high temperature, a majority of synMuv B mutants irreversibly arrest at the L1 stage. High temperature arrest (HTA) is accompanied by upregulation of many genes characteristic of germ line, including genes encoding components of the synaptonemal complex and other meiosis proteins. HTA is suppressed by loss of global regulators of germline chromatin, including MES-4, MRG-1, ISW-1 and the MES-2/3/6 complex, revealing that arrest is caused by somatic cells possessing a germline-like chromatin state. Germline genes are preferentially misregulated in the intestine, and necessity and sufficiency tests demonstrate that the intestine is the tissue responsible for HTA. We propose that synMuv B mutants fail to erase or antagonize an inherited germline chromatin state in somatic cells during embryonic and early larval development. As a consequence, somatic cells gain a germline program of gene expression in addition to their somatic program, leading to a mixed fate. Somatic expression of germline genes is enhanced at elevated temperature, leading to developmentally compromised somatic cells and arrest of newly hatched larvae.

Control of Oocyte Growth and Meiotic Maturation in Caenorhabditis elegans

In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. Chromosome segregation errors in female meiosis I are the leading cause of human birth defects, and age-related changes in the hormonal environment of the ovary are a suggested cause. Caenorhabditis elegans is emerging as a genetic paradigm for studying hormonal control of meiotic maturation. The meiotic maturation processes in C. elegans and mammals share a number of biological and molecular similarities. Major sperm protein (MSP) and luteinizing hormone (LH), though unrelated in sequence, both trigger meiotic resumption using somatic Gα(s)-adenylate cyclase pathways and soma-germline gap-junctional communication. At a molecular level, the oocyte responses apparently involve the control of conserved protein kinase pathways and post-transcriptional gene regulation in the oocyte. At a cellular level, the responses include cortical cytoskeletal rearrangement, nuclear envelope breakdown, assembly of the acentriolar meiotic spindle, chromosome segregation, and likely changes important for fertilization and the oocyte-to-embryo transition. This chapter focuses on signaling mechanisms required for oocyte growth and meiotic maturation in C. elegans and discusses how these mechanisms coordinate the completion of meiosis and the oocyte-to-embryo transition.

DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells.

P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs).

Analysis of smu-1, a gene that regulates the alternative splicing of unc-52 pre-mRNA in Caenorhabditis elegans.

ABSTRACT Mutations in the smu-1 gene of Caenorhabditis elegans were previously shown to suppress mutations in the genes mec-8 and unc-52.mec-8 encodes a putative RNA binding protein that affects the accumulation of specific alternatively spliced mRNA isoforms produced by unc-52 and other genes.unc-52 encodes a set of basement membrane proteins, homologs of mammalian perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis, and cuticle. We show that a presumptive null mutation in smu-1suppresses nonsense mutations in exon 17 but not exon 18 ofunc-52 and enhances the phenotype conferred by anunc-52 splice site mutation in intron 16. We have used reverse transcription-PCR and RNase protection to show that loss-of-function smu-1 mutations enhance accumulation in larvae of an alternatively spliced isoform that skips exon 17 but not exon 18 of unc-52. We have identified smu-1 molecularly; it encodes a nuclearly localized protein that contains five WD motifs and is ubiquitously expressed. The SMU-1 amino acid sequence is more than 60% identical to a predicted human protein of unknown function. We propose that smu-1 encodes a trans-acting factor that regulates the alternative splicing of the pre-mRNA ofunc-52 and other genes.

The TRIM-NHL Protein LIN-41 and the OMA RNA-Binding Proteins Antagonistically Control the Prophase-to-Metaphase Transition and Growth of Caenorhabditis elegans Oocytes

In many animals, oocytes enter meiosis early in their development but arrest in meiotic prophase I. Oocyte growth, which occurs during this arrest period, enables the acquisition of meiotic competence and the capacity to produce healthy progeny. Meiotic resumption, or meiotic maturation, involves the transition to metaphase I (M phase) and is regulated by intercellular signaling and cyclin-dependent kinase activation. Premature meiotic maturation would be predicted to diminish fertility as the timing of this event, which normally occurs after oocyte growth is complete, is crucial. In the accompanying article in this issue, we identify the highly conserved TRIM-NHL protein LIN-41 as a translational repressor that copurifies with OMA-1 and OMA-2, RNA-binding proteins redundantly required for normal oocyte growth and meiotic maturation. In this article, we show that LIN-41 enables the production of high-quality oocytes and plays an essential role in controlling and coordinating oocyte growth and meiotic maturation. lin-41 null mutants display a striking defect that is specific to oogenesis: pachytene-stage cells cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, assemble spindles, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 appears to contribute to premature M-phase entry in lin-41 mutant oocytes. Genetic and phenotypic analyses indicate that LIN-41 and OMA-1/2 exhibit an antagonistic relationship, and we suggest that translational regulation by these proteins could be important for controlling and coordinating oocyte growth and meiotic maturation.

Translational Control of the Oogenic Program by Components of OMA Ribonucleoprotein Particles in Caenorhabditis elegans

The oocytes of most sexually reproducing animals arrest in meiotic prophase I. Oocyte growth, which occurs during this period of arrest, enables oocytes to acquire the cytoplasmic components needed to produce healthy progeny and to gain competence to complete meiosis. In the nematode Caenorhabditis elegans, the major sperm protein hormone promotes meiotic resumption (also called meiotic maturation) and the cytoplasmic flows that drive oocyte growth. Prior work established that two related TIS11 zinc-finger RNA-binding proteins, OMA-1 and OMA-2, are redundantly required for normal oocyte growth and meiotic maturation. We affinity purified OMA-1 and identified associated mRNAs and proteins using genome-wide expression data and mass spectrometry, respectively. As a class, mRNAs enriched in OMA-1 ribonucleoprotein particles (OMA RNPs) have reproductive functions. Several of these mRNAs were tested and found to be targets of OMA-1/2-mediated translational repression, dependent on sequences in their 3′-untranslated regions (3′-UTRs). Consistent with a major role for OMA-1 and OMA-2 in regulating translation, OMA-1-associated proteins include translational repressors and activators, and some of these proteins bind directly to OMA-1 in yeast two-hybrid assays, including OMA-2. We show that the highly conserved TRIM-NHL protein LIN-41 is an OMA-1-associated protein, which also represses the translation of several OMA-1/2 target mRNAs. In the accompanying article in this issue, we show that LIN-41 prevents meiotic maturation and promotes oocyte growth in opposition to OMA-1/2. Taken together, these data support a model in which the conserved regulators of mRNA translation LIN-41 and OMA-1/2 coordinately control oocyte growth and the proper spatial and temporal execution of the meiotic maturation decision.

Regulation of maternal Wnt mRNA translation in C. elegans embryos

The restricted spatiotemporal translation of maternal mRNAs, which is crucial for correct cell fate specification in early C. elegans embryos, is regulated primarily through the 3′UTR. Although genetic screens have identified many maternally expressed cell fate-controlling RNA-binding proteins (RBPs), their in vivo targets and the mechanism(s) by which they regulate these targets are less clear. These RBPs are translated in oocytes and localize to one or a few blastomeres in a spatially and temporally dynamic fashion unique for each protein and each blastomere. Here, we characterize the translational regulation of maternally supplied mom-2 mRNA, which encodes a Wnt ligand essential for two separate cell-cell interactions in early embryos. A GFP reporter that includes only the mom-2 3′UTR is translationally repressed properly in oocytes and early embryos, and then correctly translated only in the known Wnt signaling cells. We show that the spatiotemporal translation pattern of this reporter is regulated combinatorially by a set of nine maternally supplied RBPs. These nine proteins all directly bind the mom-2 3′UTR in vitro and function as positive or negative regulators of mom-2 translation in vivo. The net translational readout for the mom-2 3′UTR reporter is determined by competitive binding between positive- and negative-acting RBPs for the 3′UTR, along with the distinct spatiotemporal localization patterns of these regulators. We propose that the 3′UTR of maternal mRNAs contains a combinatorial code that determines the topography of associated RBPs, integrating positive and negative translational inputs.

Germ plasm: Protein degradation in the soma

Germ plasm is a specialized cytoplasm that is physically segregated to the germline cells during early embryogenesis. Recent results suggest that, in Caenorhabditis elegans, germ plasm is also prevented from accumulating in somatic lineages by a ubiquitin ligase that targets germ plasm proteins for degradation.

Lin-41 and oma ribonucleoprotein complexes mediate a translational repression-to-activation switch controlling oocyte meiotic maturation and the oocyte-to-embryo transition in caenorhabditis elegans

An extended meiotic prophase is a hallmark of oogenesis. Hormonal signaling activates the CDK1/cyclin B kinase to promote oocyte meiotic maturation, which involves nuclear and cytoplasmic events. Nuclear maturation encompasses nuclear envelope breakdown, meiotic spindle assembly, and chromosome segregation. Cytoplasmic maturation involves major changes in oocyte protein translation and cytoplasmic organelles and is poorly understood. In the nematode Caenorhabditis elegans, sperm release the major sperm protein (MSP) hormone to promote oocyte growth and meiotic maturation. Large translational regulatory ribonucleoprotein (RNP) complexes containing the RNA-binding proteins OMA-1, OMA-2, and LIN-41 regulate meiotic maturation downstream of MSP signaling. To understand the control of translation during meiotic maturation, we purified LIN-41-containing RNPs and characterized their protein and RNA components. Protein constituents of LIN-41 RNPs include essential RNA-binding proteins, the GLD-2 cytoplasmic poly(A) polymerase, the CCR4-NOT deadenylase complex, and translation initiation factors. RNA sequencing defined messenger RNAs (mRNAs) associated with both LIN-41 and OMA-1, as well as sets of mRNAs associated with either LIN-41 or OMA-1. Genetic and genomic evidence suggests that GLD-2, which is a component of LIN-41 RNPs, stimulates the efficient translation of many LIN-41-associated transcripts. We analyzed the translational regulation of two transcripts specifically associated with LIN-41 which encode the RNA regulators SPN-4 and MEG-1. We found that LIN-41 represses translation of spn-4 and meg-1, whereas OMA-1 and OMA-2 promote their expression. Upon their synthesis, SPN-4 and MEG-1 assemble into LIN-41 RNPs prior to their functions in the embryo. This study defines a translational repression-to-activation switch as a key element of cytoplasmic maturation.

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

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.