Christian R. Eckmann

Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation

P Granule Conundrum In many organisms, the presumptive germ cells can be distinguished from somatic cells by the presence of distinctive cytoplasmic granules. In Caenorhabditis elegans, these P granules are more or less uniformly distributed in the oocyte and one-cell stage of the fertilized egg. By the end of the first cleavage, however, the anterior cell is essentially free of P granules, whereas the posterior cell still displays a prominent population of granules. Exactly how this process occurs and whether it involves directed migration of the granules is unclear. Now Brangwynne et al. (p. 1729, published online 21 May; see the Perspective by Le Goff and Lecuit) provide evidence that localization occurs by a quite different mechanism, controlled dissolution and condensation of granule components. This type of cytoplasmic remodeling by physicochemical mechanisms can now be looked for in other cellular and developmental systems. Localization of RNA and protein-rich germ-cell granules occurs by controlled dissolution and condensation. In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs. In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo. Localization of P granules and their physical nature remain poorly understood. Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension. As with other liquids, P granules rapidly dissolved and condensed. Localization occurred by a biased increase in P granule condensation at the posterior. This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell. Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm.

A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans.

Messenger RNA regulation is a critical mode of controlling gene expression. Regulation of mRNA stability and translation is linked to controls of poly(A) tail length. Poly(A) lengthening can stabilize and translationally activate mRNAs, whereas poly(A) removal can trigger degradation and translational repression. Germline granules (for example, polar granules in flies, P granules in worms) are ribonucleoprotein particles implicated in translational control. Here we report that the Caenorhabditis elegans gene gld-2, a regulator of mitosis/meiosis decision and other germline events, encodes the catalytic moiety of a cytoplasmic poly(A) polymerase (PAP) that is associated with P granules in early embryos. Importantly, the GLD-2 protein sequence has diverged substantially from that of conventional eukaryotic PAPs, and lacks a recognizable RRM (RNA recognition motif)-like domain. GLD-2 has little PAP activity on its own, but is stimulated in vitro by GLD-3. GLD-3 is also a developmental regulator, and belongs to the Bicaudal-C family of RNA binding proteins. We suggest that GLD-2 is the prototype for a class of regulatory cytoplasmic PAPs that are recruited to specific mRNAs by a binding partner, thereby targeting those mRNAs for polyadenylation and increased expression.

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

Significance Phase transitions have recently emerged as a key mechanism for intracellular organization. However, the underlying molecular interactions and nature of the resulting condensed phases are poorly understood. Here, we identify a role for LAF-1 in the liquid phase separation of P granules—RNA/protein assemblies implicated in germ-line maintenance. We adapt microrheology techniques to measure precise viscoelastic properties of LAF-1 liquid droplets. Our experiments reveal that electrostatic disordered protein interactions give rise to droplets with tunable material properties. RNA can fluidize protein droplets by decreasing the viscosity and increasing internal molecular dynamics. Our results provide insight into the mechanism by which molecular level interactions can give rise to liquid phase organelles with tunable material properties, potentially underlying biologically adaptable functions. 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.

Mouse HORMAD1 and HORMAD2, Two Conserved Meiotic Chromosomal Proteins, Are Depleted from Synapsed Chromosome Axes with the Help of TRIP13 AAA-ATPase

Meiotic crossovers are produced when programmed double-strand breaks (DSBs) are repaired by recombination from homologous chromosomes (homologues). In a wide variety of organisms, meiotic HORMA-domain proteins are required to direct DSB repair towards homologues. This inter-homologue bias is required for efficient homology search, homologue alignment, and crossover formation. HORMA-domain proteins are also implicated in other processes related to crossover formation, including DSB formation, inhibition of promiscuous formation of the synaptonemal complex (SC), and the meiotic prophase checkpoint that monitors both DSB processing and SCs. We examined the behavior of two previously uncharacterized meiosis-specific mouse HORMA-domain proteins—HORMAD1 and HORMAD2—in wild-type mice and in mutants defective in DSB processing or SC formation. HORMADs are preferentially associated with unsynapsed chromosome axes throughout meiotic prophase. We observe a strong negative correlation between SC formation and presence of HORMADs on axes, and a positive correlation between the presumptive sites of high checkpoint-kinase ATR activity and hyper-accumulation of HORMADs on axes. HORMADs are not depleted from chromosomes in mutants that lack SCs. In contrast, DSB formation and DSB repair are not absolutely required for depletion of HORMADs from synapsed axes. A simple interpretation of these findings is that SC formation directly or indirectly promotes depletion of HORMADs from chromosome axes. We also find that TRIP13 protein is required for reciprocal distribution of HORMADs and the SYCP1/SC-component along chromosome axes. Similarities in mouse and budding yeast meiosis suggest that TRIP13/Pch2 proteins have a conserved role in establishing mutually exclusive HORMAD-rich and synapsed chromatin domains in both mouse and yeast. Taken together, our observations raise the possibility that involvement of meiotic HORMA-domain proteins in the regulation of homologue interactions is conserved in mammals.

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. WIREs RNA 2011 2 348–361 DOI: 10.1002/wrna.56

The GLD-2 poly(A) polymerase activates gld-1 mRNA in the Caenorhabditis elegans germ line

mRNA regulation is crucial for many aspects of metazoan development and physiology, including regulation of stem cells and synaptic plasticity. In the nematode germ line, RNA regulators control stem cell maintenance, the sperm/oocyte decision, and progression through meiosis. Of particular importance to this work are three GLD (germ-line development) regulatory proteins, each of which promotes entry into the meiotic cell cycle: GLD-1 is a STAR/Quaking translational repressor, GLD-2 is a cytoplasmic poly(A) polymerase, and GLD-3 is a homolog of Bicaudal-C. Here we report that the gld-1 mRNA is a direct target of the GLD-2 poly(A) polymerase: polyadenylation of gld-1 mRNA depends on GLD-2, the abundance of GLD-1 protein is dependent on GLD-2, and the gld-1 mRNA coimmunoprecipitates with both GLD-2 and GLD-3 proteins. We suggest that the GLD-2 poly(A) polymerase enhances entry into the meiotic cell cycle at least in part by activating GLD-1 expression. The importance of this conclusion is twofold. First, the activation of gld-1 mRNA by GLD-2 identifies a positive regulatory step that reinforces the decision to enter the meiotic cell cycle. Second, gld-1 mRNA is initially repressed by FBF (for fem-3 binding factor) to maintain stem cells but then becomes activated by the GLD-2 poly(A) polymerase once stem cells begin to make the transition into the meiotic cell cycle. Therefore, a molecular switch regulates gld-1 mRNA activity to accomplish the transition from mitosis to meiosis.

Two conserved regulatory cytoplasmic poly(A) polymerases, GLD-4 and GLD-2, regulate meiotic progression in C. elegans

Translational regulation is heavily employed during developmental processes to control the timely accumulation of proteins independently of gene transcription. In particular, mRNA poly(A) tail metabolism in the cytoplasm is a key determinant for balancing an mRNAs translational output and its decay rate. Noncanonical poly(A) polymerases (PAPs), such as germline development defective-2 (GLD-2), can mediate poly(A) tail extension. Little is known about the regulation and functional complexity of cytoplasmic PAPs. Here we report the discovery of Caenorhabditis elegans GLD-4, a cytoplasmic PAP present in P granules that is orthologous to Trf4/5p from budding yeast. GLD-4 enzymatic activity is enhanced by its interaction with GLS-1, a protein associated with the RNA-binding protein GLD-3. GLD-4 is predominantly expressed in germ cells, and its activity is essential for early meiotic progression of male and female gametes in the absence of GLD-2. For commitment into female meiosis, both PAPs converge on at least one common target mRNA-i.e., gld-1 mRNA-and, as a consequence, counteract the repressive action of two PUF proteins and the putative deadenylase CCR-4. Together our findings suggest that two different cytoplasmic PAPs stabilize and translationally activate several meiotic mRNAs to provide a strong fail-safe mechanism for early meiotic progression.

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

Summary 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.

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.

GLS-1, a Novel P Granule Component, Modulates a Network of Conserved RNA Regulators to Influence Germ Cell Fate Decisions

Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.