Germline immortality relies on TRIM32-mediated turnover of a maternal mRNA activator in C. elegans.

How the germ line achieves a clean transition from maternal to zygotic gene expression control is a fundamental problem in sexually reproducing organisms. Whereas several mechanisms terminate the maternal program in the soma, this combined molecular reset and handover are poorly understood for primordial germ cells (PGCs). Here, we show that GRIF-1, a TRIM32-related and presumed E3 ubiquitin ligase in Caenorhabditis elegans, eliminates the maternal cytoplasmic poly(A) polymerase (cytoPAP) complex by targeting the germline-specific intrinsically disordered region of its enzymatic subunit, GLD-2, for proteasome-mediated degradation. Interference with cytoPAP turnover in PGCs causes frequent transgenerational sterility and, eventually, germline mortality. Hence, positively acting maternal RNA regulators are cleared via the proteasome system to avoid likely interference between maternal and zygotic gene expression programs to maintain transgenerational fertility and acquire germline immortality. This strategy is likely used in all animals that preform their immortal germ line via maternally inherited germplasm determinants.

Stage-specific combinations of opposing poly(A) modifying enzymes guide gene expression during early oogenesis.

RNA-modifying enzymes targeting mRNA poly(A) tails are universal regulators of post-transcriptional gene expression programs. Current data suggest that an RNA-binding protein (RBP) directed tug-of-war between tail shortening and re-elongating enzymes operates in the cytoplasm to repress or activate specific mRNA targets. While this concept is widely accepted, it was primarily described in the final meiotic stages of frog oogenesis and relies molecularly on a single class of RBPs, i.e. CPEBs, the deadenylase PARN and cytoplasmic poly(A) polymerase GLD-2. Using the spatial and temporal resolution of female gametogenesis in the nematode C. elegans, we determined the distinct roles of known deadenylases throughout germ cell development and discovered that the Ccr4-Not complex is the main antagonist to GLD-2-mediated mRNA regulation. We find that the Ccr4-Not/GLD-2 balance is critical for essentially all steps of oocyte production and reiteratively employed by various classes of RBPs. Interestingly, its two deadenylase subunits appear to affect mRNAs stage specifically: while a Caf1/GLD-2 antagonism regulates mRNA abundance during all stages of oocyte production, a Ccr4/GLD-2 antagonism regulates oogenesis in an mRNA abundance independent manner. Our combined data suggests that the Ccr4-Not complex represents the evolutionarily conserved molecular opponent to GLD-2 providing an antagonistic framework of gene-specific poly(A)-tail regulation.

MAPK signaling couples SCF-mediated degradation of translational regulators to oocyte meiotic progression.

RNA-binding proteins (RBPs) are important regulators of gene expression programs, especially during gametogenesis. How the abundance of particular RBPs is restricted to defined stages of meiosis remains largely elusive. Here, we report a molecular pathway that subjects two nonrelated but broadly evolutionarily conserved translational regulators (CPB-3/CPEB and GLD-1/STAR) to proteosomal degradation in Caenorhabditis elegans germ cells at the transition from pachytene to diplotene of meiotic prophase. Both RBPs are recognized by the same ubiquitin ligase complex, containing the molecular scaffold Cullin-1 and the tumor suppressor SEL-10/FBXW7 as its substrate recognition subunit. Destabilization of either RBP through this Skp, Cullin, F-box-containing complex (SCF) ubiquitin ligase appears to loosen its negative control over established target mRNAs, and presumably depends on a prior phosphorylation of CPB-3 and GLD-1 by MAPK (MPK-1), whose activity increases in mid- to late pachytene to promote meiotic progression and oocyte differentiation. Thus, we propose that the orchestrated degradation of RBPs via MAPK-signaling cascades during germ cell development may act to synchronize meiotic with sexual differentiation gene expression changes.

Polyadenylation is the key aspect of GLD-2 function in C. elegans.

The role of many enzymes extends beyond their dedicated catalytic activity by fulfilling important cellular functions in a catalysis-independent fashion. In this aspect, little is known about 3'-end RNA-modifying enzymes that belong to the class of nucleotidyl transferases. Among these are noncanonical poly(A) polymerases, a group of evolutionarily conserved enzymes that are critical for gene expression regulation, by adding adenosines to the 3'-end of RNA targets. In this study, we investigate whether the functions of the cytoplasmic poly(A) polymerase (cytoPAP) GLD-2 in C. elegans germ cells exclusively depend on its catalytic activity. To this end, we analyzed a specific missense mutation affecting a conserved amino acid in the catalytic region of GLD-2 cytoPAP. Although this mutated protein is expressed to wild-type levels and incorporated into cytoPAP complexes, we found that it cannot elongate mRNA poly(A) tails efficiently or promote GLD-2 target mRNA abundance. Furthermore, germ cell defects in animals expressing this mutant protein strongly resemble those lacking the GLD-2 protein altogether, arguing that only the polyadenylation activity of GLD-2 is essential for gametogenesis. In summary, we propose that all known molecular and biological functions of GLD-2 depend on its enzymatic activity, demonstrating that polyadenylation is the key mechanism of GLD-2 functionality. Our findings highlight the enzymatic importance of noncanonical poly(A) polymerases and emphasize the pivotal role of poly(A) tail-centered cytoplasmic mRNA regulation in germ cell biology.

Polar Positioning of Phase-Separated Liquid Compartments in Cells Regulated by an mRNA Competition Mechanism.

P granules are non-membrane-bound RNA-protein compartments that are involved in germline development in C. elegans. They are liquids that condense at one end of the embryo by localized phase separation, driven by gradients of polarity proteins such as the mRNA-binding protein MEX-5. To probe how polarity proteins regulate phase separation, we combined biochemistry and theoretical modeling. We reconstitute P granule-like droplets in vitro using a single protein PGL-3. By combining in vitro reconstitution with measurements of intracellular concentrations, we show that competition between PGL-3 and MEX-5 for mRNA can regulate the formation of PGL-3 droplets. Using theory, we show that, in a MEX-5 gradient, this mRNA competition mechanism can drive a gradient of P granule assembly with similar spatial and temporal characteristics to P granule assembly in vivo. We conclude that gradients of polarity proteins can position RNP granules during development by using RNA competition to regulate local phase separation.

Structural basis for the antagonistic roles of RNP-8 and GLD-3 in GLD-2 poly(A)-polymerase activity.

Cytoplasmic polyadenylation drives the translational activation of specific mRNAs in early metazoan development and is performed by distinct complexes that share the same catalytic poly(A)-polymerase subunit, GLD-2. The activity and specificity of GLD-2 depend on its binding partners. In Caenorhabditis elegans, GLD-2 promotes spermatogenesis when bound to GLD-3 and oogenesis when bound to RNP-8. GLD-3 and RNP-8 antagonize each other and compete for GLD-2 binding. Following up on our previous mechanistic studies of GLD-2-GLD-3, we report here the 2.5 Å resolution structure and biochemical characterization of a GLD-2-RNP-8 core complex. In the structure, RNP-8 embraces the poly(A)-polymerase, docking onto several conserved hydrophobic hotspots present on the GLD-2 surface. RNP-8 stabilizes GLD-2 and indirectly stimulates polyadenylation. RNP-8 has a different amino-acid sequence and structure as compared to GLD-3. Yet, it binds the same surfaces of GLD-2 by forming alternative interactions, rationalizing the remarkable versatility of GLD-2 complexes.

Translational activation maintains germline tissue homeostasis during adulthood.

Adult tissue maintenance is achieved through a tightly controlled equilibrium of 2 opposing cell fates: stem cell proliferation and differentiation. In recent years, the germ line emerged as a powerful in vivo model tissue to investigate the underlying gene expression mechanisms regulating this balance. Studies in numerous organisms highlighted the prevalence of post-transcriptional mRNA regulation, which relies on RNA-targeting factors that influence mRNA fates (e.g. decay or translational efficiency). Conserved translational repressors were identified that build negative feedback loops to ensure one or the other cell fate. However, to facilitate a fast and efficient transition between 2 opposing cell fates, translational repression per se appears not to be sufficient, suggesting the involvement of additional modes of gene expression regulation. Cytoplasmic poly(A) polymerases (cytoPAPs) represent a unique class of post-transcriptional mRNA regulators that modify mRNA 3' ends and positively influence cytoplasmic mRNA fates. We recently discovered that the 2 main cytoPAPs, GLD-2 and GLD-4, use distinct mechanisms to promote gene expression and that cytoPAP-mediated mRNA activation is important for regulating the size of the proliferative germ cell pool in the adult Caenorhabditis elegans gonad. Here, we comment on the different mechanisms of the 2 cytoPAPs as translational activators in germ cell development and focus on their biological roles in maintaining the balance between germline stem cell proliferation and differentiation in the Caenorhabditis elegans gonad.

Structural basis for the activation of the C. elegans noncanonical cytoplasmic poly(A)-polymerase GLD-2 by GLD-3.

The Caenorhabditis elegans germ-line development defective (GLD)-2-GLD-3 complex up-regulates the expression of genes required for meiotic progression. GLD-2-GLD-3 acts by extending the short poly(A) tail of germ-line-specific mRNAs, switching them from a dormant state into a translationally active state. GLD-2 is a cytoplasmic noncanonical poly(A) polymerase that lacks the RNA-binding domain typical of the canonical nuclear poly(A)-polymerase Pap1. The activity of C. elegans GLD-2 in vivo and in vitro depends on its association with the multi-K homology (KH) domain-containing protein, GLD-3, a homolog of Bicaudal-C. We have identified a minimal polyadenylation complex that includes the conserved nucleotidyl-transferase core of GLD-2 and the N-terminal domain of GLD-3, and determined its structure at 2.3-Å resolution. The structure shows that the N-terminal domain of GLD-3 does not fold into the predicted KH domain but wraps around the catalytic domain of GLD-2. The picture that emerges from the structural and biochemical data are that GLD-3 activates GLD-2 both indirectly by stabilizing the enzyme and directly by contributing positively charged residues near the RNA-binding cleft. The RNA-binding cleft of GLD-2 has distinct structural features compared with the poly(A)-polymerases Pap1 and Trf4. Consistently, GLD-2 has distinct biochemical properties: It displays unusual specificity in vitro for single-stranded RNAs with at least one adenosine at the 3' end. GLD-2 thus appears to have evolved specialized nucleotidyl-transferase properties that match the 3' end features of dormant cytoplasmic mRNAs.

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA-protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA-protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.

GLD-4-mediated translational activation regulates the size of the proliferative germ cell pool in the adult C. elegans germ line.

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.

The cytoplasmic poly(A) polymerases GLD-2 and GLD-4 promote general gene expression via distinct mechanisms.

Post-transcriptional gene regulation mechanisms decide on cellular mRNA activities. Essential gatekeepers of post-transcriptional mRNA regulation are broadly conserved mRNA-modifying enzymes, such as cytoplasmic poly(A) polymerases (cytoPAPs). Although these non-canonical nucleotidyltransferases efficiently elongate mRNA poly(A) tails in artificial tethering assays, we still know little about their global impact on poly(A) metabolism and their individual molecular roles in promoting protein production in organisms. Here, we use the animal model Caenorhabditis elegans to investigate the global mechanisms of two germline-enriched cytoPAPs, GLD-2 and GLD-4, by combining polysome profiling with RNA sequencing. Our analyses suggest that GLD-2 activity mediates mRNA stability of many translationally repressed mRNAs. This correlates with a general shortening of long poly(A) tails in gld-2-compromised animals, suggesting that most if not all targets are stabilized via robust GLD-2-mediated polyadenylation. By contrast, only mild polyadenylation defects are found in gld-4-compromised animals and few mRNAs change in abundance. Interestingly, we detect a reduced number of polysomes in gld-4 mutants and GLD-4 protein co-sediments with polysomes, which together suggest that GLD-4 might stimulate or maintain translation directly. Our combined data show that distinct cytoPAPs employ different RNA-regulatory mechanisms to promote gene expression, offering new insights into translational activation of mRNAs.

Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs.

Poly(A) tail length is a readout of an mRNA's translatability and stability, especially in developmental systems. PolyAdenylation Test (PAT) assays attempt to quickly measure the average poly(A) tail length of RNAs of experimental interest. Here we present sPAT, splint-mediated PAT, a procedure that uses a DNA splint to aid in the ligation of an RNA-tag to the poly(A) tail of an mRNA. In comparison to other PAT methodologies, including ePAT, sPAT is highly sensitive to low-abundance mRNAs, gives a more accurate profile of the poly(A) tail distribution, and requires little starting material. To demonstrate its strength, we calibrated sPAT on defined poly(A) tails of synthetic mRNAs, reassessed developmentally regulated poly(A) tail-length changes of known mRNAs from established model organisms, and extended it to the emerging evolutionary developmental nematode model Pristionchus pacificus. Lastly, we used sPAT to analyze the contribution of the two cytoplasmic poly(A) polymerases GLD-2 and GLD-4, and the deadenylase CCR-4, onto Caenorhabditis elegans gld-1 mRNA that encodes a translationally controlled tumor suppressor whose poly(A) tail length measurement proved elusive.

The Ccr4-Not deadenylase complex constitutes the main poly(A) removal activity in C. elegans.

Post-transcriptional regulatory mechanisms are widely used to control gene expression programs of tissue development and physiology. Controlled 3' poly(A) tail-length changes of mRNAs provide a mechanistic basis of such regulation, affecting mRNA stability and translational competence. Deadenylases are a conserved class of enzymes that facilitate poly(A) tail removal, and their biochemical activities have been mainly studied in the context of single-cell systems. Little is known about the different deadenylases and their biological role in multicellular organisms. In this study, we identify and characterize all known deadenylases of Caenorhabditis elegans, and identify the germ line as tissue that depends strongly on deadenylase activity. Most deadenylases are required for hermaphrodite fertility, albeit to different degrees. Whereas ccr-4 and ccf-1 deadenylases promote germline function under physiological conditions, panl-2 and parn-1 deadenylases are only required under heat-stress conditions. We also show that the Ccr4-Not core complex in nematodes is composed of the two catalytic subunits CCR-4 and CCF-1 and the structural subunit NTL-1, which we find to regulate the stability of CCF-1. Using bulk poly(A) tail measurements with nucleotide resolution, we detect strong deadenylation defects of mRNAs at the global level only in the absence of ccr-4, ccf-1 and ntl-1, but not of panl-2, parn-1 and parn-2. Taken together, this study suggests that the Ccr4-Not complex is the main deadenylase complex in C. elegans germ cells. On the basis of this and as a result of evidence in flies, we propose that the conserved Ccr4-Not complex is an essential component in post-transcriptional regulatory networks promoting animal reproduction.

Fertility and germline stem cell maintenance under different diets requires nhr-114/HNF4 in C. elegans.

Animals can thrive on variable food resources as a result of autonomous processes and beneficial relationships with their gut microbes [1]. Food intake elicits major physiological changes, which are counteracted by transient systemic responses that maintain homeostasis in the organism. This integration of external information occurs through cellular sensory elements, such as nuclear receptors, which modulate gene expression in response to specific cues [2]. Given the importance of germline stem cells (GSCs) for the development of the germline and the continuity of species, it is reasonable to assume that GSCs might be shielded from the negative influence of environmental perturbations. To our knowledge, however, there are no mechanisms reported that protect GSCs from harmful dietary metabolites. Using Caenorhabditis elegans as a model, we report that the somatic activity of the conserved nuclear receptor nhr-114/HNF4 protects GSC integrity from dietary metabolites. In the absence of nhr-114 and on certain bacterial diets, otherwise somatically normal animals accumulate germ cell division defects during development and become sterile. We found that, in nhr-114(-) animals, the induction of germline defects and sterility depend on bacterial metabolic status, with respect to the essential amino acid tryptophan. This illustrates an animal-microbe interaction in which somatic nuclear receptor activity preserves the germline by buffering against dietary metabolites, most likely through a somatic detoxifying response. Overall, our findings uncover an unprecedented, and presumably evolutionarily conserved, soma-to-germline axis of communication that maintains reproductive robustness on variable food resources.

Mind the gut: Dietary impact on germline stem cells and fertility.

Animals thrive in environments where food resources are abundant. While this correlation between population growth and food abundance is well established, much less is known about the influence of diet quality on physiological and developmental programs that support animal reproduction. Here we discuss dietary impact on fertility, and highlight a recent report on the activity of a nuclear receptor that protects against dietary metabolites to maintain germline stem cell integrity and reproduction.

Translational control in the Caenorhabditis elegans germ line.

Translational control is a prevalent form of gene expression regulation in the Caenorhabditis elegans germ line. Linking the amount of protein synthesis to mRNA quantity and translational accessibility in the cell cytoplasm provides unique advantages over DNA-based controls for developing germ cells. This mode of gene expression is especially exploited in germ cell fate decisions and during oogenesis, when the developing oocytes stockpile hundreds of different mRNAs required for early embryogenesis. Consequently, a dense web of RNA regulators, consisting of diverse RNA-binding proteins and RNA-modifying enzymes, control the translatability of entire mRNA expression programs. These RNA regulatory networks are tightly coupled to germ cell developmental progression and are themselves under translational control. The underlying molecular mechanisms and RNA codes embedded in the mRNA molecules are beginning to be understood. Hence, the C. elegans germ line offers fertile grounds for discovering post-transcriptional mRNA regulatory mechanisms and emerges as great model for a systems level understanding of translational control during development.

Subcellular specialization of multifaceted 3'end modifying nucleotidyltransferases.

While canonical 3'end modifications of mRNAs or tRNAs are well established, recent technological advances in RNA analysis have given us a glimpse of how widespread other types of distinctive 3'end modifications appear to be. Next to alternative nuclear or cytoplasmic polyadenylation mechanisms, evidence accumulated for a variety of 3'end mono-nucleotide and oligo-nucleotide additions of primarily adenosines or uracils on a variety of RNA species. Enzymes responsible for such non-templated additions are non-canonical RNA nucleotidyltransferases, which possess surprising flexibility in RNA substrate selection and enzymatic activity. We will highlight recent findings supporting the view that RNA nucleotidyltransferase activity, RNA target selection and sub-compartimentalization are spatially, temporally and physiologically regulated by dedicated co-factors. Along with the diversification of non-coding RNA classes, the evolutionary conservation of these multifaceted RNA modifiers underscores the prevalence and importance of diverse 3'end formation mechanisms.

Control of poly(A) tail length.

Poly(A) tails have long been known as stable 3' modifications of eukaryotic mRNAs, added during nuclear pre-mRNA processing. It is now appreciated that this modification is much more diverse: A whole new family of poly(A) polymerases has been discovered, and poly(A) tails occur as transient destabilizing additions to a wide range of different RNA substrates. We review the field from the perspective of poly(A) tail length. Length control is important because (1) poly(A) tail shortening from a defined starting point acts as a timer of mRNA stability, (2) changes in poly(A) tail length are used for the purpose of translational regulation, and (3) length may be the key feature distinguishing between the stabilizing poly(A) tails of mRNAs and the destabilizing oligo(A) tails of different unstable RNAs. The mechanism of length control during nuclear processing of pre-mRNAs is relatively well understood and is based on the changes in the processivity of poly(A) polymerase induced by two RNA-binding proteins. Developmentally regulated poly(A) tail extension also generates defined tails; however, although many of the proteins responsible are known, the reaction is not understood mechanistically. Finally, destabilizing oligoadenylation does not appear to have inherent length control. Rather, average tail length results from the balance between polyadenylation and deadenylation.

Four KH domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular structural platform.

Caenorhabditis elegans GLD-3 is a five K homology (KH) domain-containing protein involved in the translational control of germline-specific mRNAs during embryogenesis. GLD-3 interacts with the cytoplasmic poly(A)-polymerase GLD-2. The two proteins cooperate to recognize target mRNAs and convert them into a polyadenylated, translationally active state. We report the 2.8-Å-resolution crystal structure of a proteolytically stable fragment encompassing the KH2, KH3, KH4, and KH5 domains of C. elegans GLD-3. The structure reveals that the four tandem KH domains are organized into a globular structural unit. The domains are involved in extensive side-by-side interactions, similar to those observed in previous structures of dimeric KH domains, as well as head-to-toe interactions. Small-angle X-ray scattering reconstructions show that the N-terminal KH domain (KH1) forms a thumb-like protrusion on the KH2-KH5 unit. Although KH domains are putative RNA-binding modules, the KH region of GLD-3 is unable in isolation to cross-link RNA. Instead, the KH1 domain mediates the direct interaction with the poly(A)-polymerase GLD-2, pointing to a function of the KH region as a protein-protein interaction platform.