Dustin L. Updike

P Granule Assembly and Function in Caenorhabditis elegans Germ Cells

Germ granules are large, non-membrane-bound, ribonucleoprotein (RNP) organelles found in the germ line cytoplasm of most, if not all, animals. The term germ granule is synonymous with the perinuclear nuage in mouse and human germ cells. These large RNPs are complexed with germ line-specific cytoplasmic structures such as the mitochondrial cloud, intermitochondrial cement, and chromatoid bodies. The widespread presence of germ granules across species and the associated germ line defects when germ granules are compromised suggest that germ granules are key determinants of the identity and special properties of germ cells. The nematode Caenorhabditis elegans has been a very fruitful model system for the study of germ granules, wherein they are referred to as P granules. P granules contain a heterogeneous mixture of RNAs and proteins. To date, most of the known germ granule proteins across species, and all of the known P granule components in C elegans, are associated with RNA metabolism, which suggests that a main function of germ granules is posttranscriptional regulation. Here we review P granule structure and localization, P granule composition, the genetic pathway of P granule assembly, and the consequences in the germ line when P granule components are lost. The findings in C elegans have important implications for the germ granule function during postnatal germ cell differentiation in mammals.

The Target of Rapamycin Pathway Antagonizes pha-4/FoxA to Control Development and Aging

BACKGROUND FoxA factors are critical regulators of embryonic development and postembryonic life, but little is know about the upstream pathways that modulate their activity. C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos and to control growth and longevity after birth. We previously identified the AAA+ ATPase homolog ruvb-1 as a potent suppressor of pha-4 mutations. RESULTS Here we show that ruvb-1 is a component of the Target of Rapamycin (TOR) pathway in C. elegans (CeTOR). Both ruvb-1 and let-363/TOR control nucleolar size and promote localization of box C/D snoRNPs to nucleoli, suggesting a role in rRNA maturation. Inactivation of let-363/TOR or ruvb-1 suppresses the lethality associated with reduced pha-4 activity. The CeTOR pathway controls protein homeostasis and also contributes to adult longevity. We find that pha-4 is required to extend adult lifespan in response to reduced CeTOR signaling. Mutations in the predicted CeTOR target rsks-1/S6 kinase or in ife-2/eIF4E also reduce protein biosynthesis and extend lifespan, but only rsks-1 mutations require pha-4 for adult longevity. In addition, rsks-1, but not ife-2, can suppress the larval lethality associated with pha-4 loss-of-function mutations. CONCLUSIONS The data suggest that pha-4 and the CeTOR pathway antagonize one another to regulate postembryonic development and adult longevity. We suggest a model in which nutrients promote TOR and S6 kinase signaling, which represses pha-4/FoxA, leading to a shorter lifespan. A similar regulatory hierarchy may function in other animals to modulate metabolism, longevity, or disease.

P granules extend the nuclear pore complex environment in the C. elegans germ line

Like the nuclear pore complex, FG repeat–containing P-granule proteins interact to help establish a size-exclusion barrier.

A Genomewide RNAi Screen for Genes That Affect the Stability, Distribution and Function of P Granules in Caenorhabditis elegans

P granules are non-membrane-bound organelles found in the germ-line cytoplasm throughout Caenorhabditis elegans development. Like their “germ granule” counterparts in other animals, P granules are thought to act as determinants of the identity and special properties of germ cells, properties that include the unique ability to give rise to all tissues of future generations of an organism. Therefore, understanding how P granules work is critical to understanding how cellular immortality and totipotency are retained, gained, and lost. Here we report on a genomewide RNAi screen in C. elegans, which identified 173 genes that affect the stability, localization, and function of P granules. Many of these genes fall into specific classes with shared P-granule phenotypes, allowing us to better understand how cellular processes such as protein degradation, translation, splicing, nuclear transport, and mRNA homeostasis converge on P-granule assembly and function. One of the more striking phenotypes is caused by the depletion of CSR-1, an Argonaute associated with an endogenous siRNA pathway that functions in the germ line. We show that CSR-1 and two other endo-siRNA pathway members, the RNA-dependent RNA polymerase EGO-1 and the helicase DRH-3, act to antagonize RNA and P-granule accumulation in the germ line. Our findings strengthen the emerging view that germ granules are involved in numerous aspects of RNA metabolism, including an endo-siRNA pathway in germ cells.

Germ-Granule Components Prevent Somatic Development in the C. elegans Germline

Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans [1]. In Drosophila, germ granules have been implicated in germline determination [2]. C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination [3], but their components are still needed for fertility [4-6]. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g., in germlines lacking MEX-3 and GLD-1 or upon neuronal induction by CHE-1 [7, 8]. However, it has not been established whether loss of P granules is the cause or effect of cell fate transformation. To test cause and effect, we severely compromised P granules by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization (GLH-1 and GLH-4) [9] and investigated whether this causes germ cells to lose totipotency and initiate somatic reprogramming. We found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate.

Contribution of the amino and carboxyl termini for PHA-4/FoxA function in Caenorhabditis elegans

FoxA transcription factors are central regulators of gut development in all animals that have been studied. Here we examine the sole Caenorhabditis elegans FoxA protein, which is called pha‐4. We describe the molecular characterization of five pha‐4 mutations and characterize their associated phenotypes. Two nonsense mutations are predicted to truncate PHA‐4 after the DNA binding domain and remove the conserved carboxyl terminus. Surprisingly, animals harboring these mutations are viable, provided the mutant mRNAs are stabilized by inactivating the nonsense‐mediated decay pathway. Two additional nonsense mutations reveal that the DNA binding domain is critical for activity. A missense mutation predicted to alter the PHA‐4 amino terminus leads to a dramatic reduction in pha‐4 activity even though the protein is expressed appropriately. We suggest that the PHA‐4 amino terminus is essential for PHA‐4 function in vivo, possibly as a transactivation domain, and can compensate for loss of the carboxyl terminus. We also provide evidence for autoregulation by PHA‐4. Developmental Dynamics 234:346–354, 2005.

CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline

Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription. SUMMARY: Germ granules in C. elegans are required for fertility and the maintenance of germ cell identity, but also function to repress sperm-specific expression in during oogenesis.

ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in caenorhabditis elegans

Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3). The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules). ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline.

A Forward Genetic Screen for Suppressors of Somatic P Granules in Caenorhabditis elegans

In Caenorhabditis elegans, germline expression programs are actively repressed in somatic tissue by components of the synMuv (synthetic multi-vulva) B chromatin remodeling complex, which include homologs of tumor suppressors Retinoblastoma (Rb/LIN-35) and Malignant Brain Tumor (MBT/LIN-61). However, the full scope of pathways that suppress germline expression in the soma is unknown. To address this, we performed a mutagenesis and screened for somatic expression of GFP-tagged PGL-1, a core P-granule nucleating protein. Eight alleles were isolated from 4000 haploid genomes. Five of these alleles exhibit a synMuv phenotype, whereas the remaining three were identified as hypomorphic alleles of known synMuv B genes, lin-13 and dpl-1. These findings suggest that most suppressors of germline programs in the soma of C. elegans are either required for viability or function through synMuv B chromatin regulation.

PQN-75 is expressed in the pharyngeal gland cells of Caenorhabditis elegans and is dispensable for germline development

ABSTRACT In Caenorhabditis elegans, five pharyngeal gland cells reside in the terminal bulb of the pharynx and extend anterior processes to five contact points in the pharyngeal lumen. Pharyngeal gland cells secrete mucin-like proteins thought to facilitate digestion, hatching, molting and assembly of the surface coat of the cuticle, but supporting evidence has been sparse. Here we show pharyngeal gland cell expression of PQN-75, a unique protein containing an N-terminal signal peptide, nucleoporin (Nup)-like phenylalanine/glycine (FG) repeats, and an extensive polyproline repeat domain with similarities to human basic salivary proline-rich pre-protein PRB2. Imaging of C-terminal tagged PQN-75 shows localization throughout pharyngeal gland cell processes but not the pharyngeal lumen; instead, aggregates of PQN-75 are occasionally found throughout the pharynx, suggesting secretion from pharyngeal gland cells into the surrounding pharyngeal muscle. PQN-75 does not affect fertility and brood size in C. elegans but confers some degree of stress resistance and thermotolerance through unknown mechanisms. Summary: PQN-75 is expressed in pharyngeal gland cells and shares similarity with human basic salivary proline-rich protein PBR2, suggesting evolutionary conservation between gland cells in the upper digestive tract.