RESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Gránulos Citoplasmáticos/metabolismo , Células Germinativas/metabolismo , Interferencia de ARN , Factores Generales de Transcripción/genética , Alelos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Hibridación Fluorescente in Situ , Microscopía Fluorescente , Mutación , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Generales de Transcripción/metabolismoRESUMEN
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.