Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

ABSTRACT

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 granules are large, non-membrane-bound, ribonucleoprotein (RNP) organelles found in the germ-line cytoplasm of most, if not all, animals (Eddy 1975; Saffman and Lasko 1999). The term "germ granule" encompasses what are known as P granules in Caenorhabditis elegans, polar granules in Drosophila melanogaster, germinal granules in Xenopus laevis, and the perinuclear nuage in mouse and human germ cells. These large RNP complexes 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 post-transcriptional regulation (Strome 2005; Seydoux and Braun 2006). Germ cells are unique in their ability to give rise to all tissues of future generations of an organism. Consequently, germ cells are considered to be both totipotent and immortal. The widespread presence of germ granules in germ cells across species and the ability of germ granule transplantation to induce functional germ cells suggest that germ granules are key determinants of the identity and special properties of germ cells (Smith 1966; Illmensee and Mahowald 1974; Ephrussi and Lehmann 1992). As more components and regulators of germ granules are identified, we will better understand their role in conferring germ cell identity and properties.

One of the earliest identified constitutive components of C. elegans P granules is PGL-1 (Kawasaki et al. 1998). Identification of genes whose loss alters the level and/or distribution of PGL-1 offers an avenue to identify other P-granule components and components that regulate P-granule assembly and stability. For example, the C. elegans VASA homolog GLH-1, another constitutive P-granule component, acts "upstream" of PGL-1; in glh-1 loss-of-function mutants, PGL-1 is not properly localized to P granules and instead a significant portion of PGL-1 is diffusely distributed in the germ-line cytoplasm (Kawasaki et al. 1998; Spike et al. 2008a). More recently another constitutive P-granule component, DEPS-1, was identified in a forward genetic screen formutants that displayPGL-1 localization defects similar to those seen in glh-1(lf) mutants (Spike et al. 2008b). Targeted studies have demonstrated that mutation or RNAi of other genes also disrupts PGL-1 in the germ line. These include genes encoding the nuclear transportins IMB-2, IMB-3, IMB-5, and IMA-3 (J. …

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