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

Abstract

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.

Fig 1. Screen for regulators of P-granule accumulation.

A) Screening strategy to isolate mutants with enlarged PGL-1::GFP granules. PGL-1::GFP worms were mutagenized, 2000 F1 progeny were cloned to individual plates, and F2 progeny were screened for homozygous (m/m) mutants with enlarged P granules B) PGL-1::GFP expression in the gonad arm of parental (P0) and mutant strains. C) Relative PGL-1 intensity in mutant germlines normalized to the parental control. D) Increased PGL-1::GFP size in mutants correlates with increased PGL-1 intensity.

Fig 2. Isolation of new CSR-1 complex alleles.

A-D) Location and flanking sequences for new alleles of CSR-1 complex components. Red lines show early stop codons, grey lines show base pair substitutions. Yellow boxes indicate a codon in the reading frame. E) Endogenous PGL-1::GFP expression in wild-type and drh-3(sam27) germlines.

Fig 3. elli-1 alleles and phenotypes.

A) Location and flanking sequences of elli-1 alleles. Red lines show early stop codons, grey lines show base pair substitutions and deletions. Yellow boxes indicate a codon in the reading frame. ELLI-1’s N-terminal predicted disordered region is shown by the green bars. B) elli-1(sam3) and elli-1(RNAi) cause bright expression of enlarged PGL-1::GFP granules. C) Fosmid rescue of bright PGL-1::GFP expression in elli-1(sam3) animals. Red body-wall muscles mark animals with the rescuing fosmid. D) Cross-section through pachytene germ cells on the surface of wild-type and elli-1(sam3) germlines stained with anti-PGL-1 (green), anti-Nuclear Pore Complex antibody mAb414 (red), and DAPI/DNA (blue). P granules in csr-1 and elli-1 germlines are enlarged and often detach from the nuclear periphery. E) Cross-section through wild-type and elli-1 germlines with the same strains as in D. Arrows show PGL-1 accumulation in the shared cytoplasm. F) GFP tagging endogenous GLH-1 in germlines of wild-type and elli-1(sam3) worms.

Fig 4. elli-1 expression.

A) Fluorescence In Situ Hybridization (FISH) of elli-1 mRNA (red) shows germline expression. DAPI/DNA is blue. B) elli-1::GFP::3xFLAG showing diffuse cytoplasmic expression with some ELLI-1 foci in a dissected germline. 4.3% of ELLI-1 foci are docked to P granules (arrowheads), but most of these foci are in the central shared cytoplasm of the rachis. C) ELLI-1::GFP partially overlaps with the expression of the P-body component CGH-1 (red), primarily in the rachis instead of at the nuclear periphery of germ cells. D) elli-1::GFP::3xFLAG expression in primordial germ cells (arrows) during embryogenesis and the first larval stage. E) Embryonic lethality associated in elli-1 worms.

Fig 5. Gene expression analysis of elli-1.

A) Volcano plot showing the fold change and significance of gene expression in elli-1. Significantly regulated genes above or below 1.2-fold are shown in red, with a subset of soma-enriched genes [43] in green (see S1 Table). B) Proportional Venn diagrams comparing overlap between the 1079 elli-1 regulated genes and previously published csr-1 and pgl-1 regulated [13], CSR-1 target [4], and soma and germline enriched [43] datasets. C) Increased average expression of CSR-1 complex and core P-granule components in the elli-1 expression array (black) and elli-1 qRTPCR (green). Pink line indicates the arbitrary 1.2-fold increased expression cutoff. Increased ego-1 and ekl-1 expression was statistically significant, but under the 1.2-fold cutoff. D) elli-1 Volcano plot showing increased expression of genes required for RNAi-dependent gene silencing (green).

Fig 6. Synthetic sterility and RNAi deficiency in elli-1.

A) Larval arrest on control (+) and his-44 (-) RNAi showing elli-1(sam3) enhances the RNAi resistant phenotype of drh-3(sam27) at permissive temperature (20°C). Same colored box plots represent duplicate experiments performed on different weeks. B) Brood sizes show that elli-1(sam3) enhances sterility (no brood) of drh-3(sam27) at permissive temperature. C) STYO14 staining of dissected germlines shows RNA pooling around the nuclear periphery and in the rachis of both csr-1 and elli-1 mutants. D) Model for CSR-1 and ELLI-1 function. P granules (yellow) reside on the nuclear periphery where they receive transcripts coming through the nuclear pore complex. The CSR-1 complex functions in P granules to recognize germline abundant or licensed transcripts. Our model is that CSR-1 and ELLI-1 function to move germline abundant or licensed transcripts through and away from P granules and into the cytoplasm. In mutants this dispersal of RNA is blocked, RNA and P granules accumulate, and fertility and RNAi efficacy is decreased. It remains unclear whether ELLI-1 directly interacts with RNA.