GLS-1, a Novel P Granule Component, Modulates a Network of Conserved RNA Regulators to Influence Germ Cell Fate Decisions ¤ Agata Rybarska, Martin Harterink , Britta Jedamzik, Adam P. Kupinski, Mark Schmid, Christian R. Eckmann* Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany Abstract 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. Citation: Rybarska A, Harterink M, Jedamzik B, Kupinski AP, Schmid M, et al. (2009) GLS-1, a Novel P Granule Component, Modulates a Network of Conserved RNA Regulators to Influence Germ Cell Fate Decisions. PLoS Genet 5(5): e1000494. doi:10.1371/journal.pgen.1000494 Editor: Anthony P. Mahowald, University of Chicago, United States of America Received January 14, 2009; Accepted April 24, 2009; Published May 22, 2009 Copyright: 2009 Rybarska et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Max Planck Society and a grant to CRE from the DFG (EC 369/1-1). MH received a stipend from the Univ. of Utrecht. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: eckmann@mpi-cbg.de ¤ Current address: Hubrecht Laboratory, Netherlands Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands Introduction Germ line and early embryonic gene expression rely largely on cytoplasmic mRNA control mechanisms, allowing for maximum flexibility of control [1]. A striking example is the unique ability of germ cells to transiently differentiate into gametes before forming a totipotent zygote upon fertilization. Many conserved cytoplasmic RNA-binding and RNA-modifying proteins have been found to support germline development, by associating with mRNA molecules in RNP complexes. In higher eukaryotes, these trans- acting factors can form larger RNP aggregates, termed germplasm granules [2–4]. Although these RNPs are anticipated to confer germ cell identity and are important for germline development their developmental regulation is largely unknown. Furthermore, it remains to be determined how these RNP complexes are utilized in an organism-specific fashion to control protein synthesis, i.e. the mRNA’s translational output. In nematodes, components of germplasm granules (P granules) are deposited maternally to the embryo and segregate to germ cell precursors, which suggests their early requirement for germline function. One such conserved maternal component is the Bicaudal-C (Bic-C) protein family member GLD-3 [5]. Bic-C proteins are involved in poly(A) tail metabolism of mRNAs [6,7]. The gld-3 locus encodes two major protein isoforms, GLD-3L and GLD-3S, of which both form a cytoplasmic poly(A) polymerase complex with GLD-2 [8]. Similar to Drosophila Bic-C, which is required for oogenesis and patterning of the embryo, GLD-3 is required for many aspects of germline development and embryogenesis, including a role in germline sex determination and germline survival [5,9,10]. The C. elegans sperm-to-oocyte switch serves as a paradigm for the analysis of post-transcriptional mRNA regulation [11]. A sex determination pathway determines the sperm and oocyte fate. Although hermaphrodites develop somatically as females, they produce a limited number of sperm during their fourth larval stage, before switching to continuous oocyte production in the adult. Therefore, the female sex determination pathway has to be temporarily suppressed to facilitate spermatogenesis. The under- PLoS Genetics | www.plosgenetics.org 1 May 2009 | Volume 5 | Issue 5 | e1000494