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

Abstract

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.

Keywords: C. elegans; CSR-1; Germ granules; Germline; Germline sex determination; P granules; Sperm; Spermatogenesis.

Fig. 1.

Comparison of wild-type and P-granule RNAi expression profiles. (A) Average germline expression of genes following control (black line) and P-granule (pink-red line, running average) RNAi (log scale). Genes are ranked from low to high expression based on the control RNAi dataset. Top box shows position of genes significantly up- or downregulated by P-granule RNAi. *RNAi targets (glh-4, pgl-3 and pgl-1, respectively). (B) Comparison of expression changes following P-granule RNAi in published enriched gene datasets (see supplementary material Table S1). Box, upper and lower quartiles; line, median; dots, mean; bars, s.d.

Fig. 2.

Comparison of wild-type and csr-1 RNAi expression profiles. (A) Average germline expression of genes following control (black line) and csr-1 (pink-red line, running average) RNAi (log scale). Genes are ranked from low to high expression, based on the control RNAi dataset. Top box shows position of genes significantly up- or downregulated by csr-1 RNAi. *RNAi target RNAi (csr-1). (B) Comparison of expression changes following csr-1 RNAi in published enriched gene datasets (see supplementary material Table S1). Box, upper and lower quartiles; line, median; dots, mean; bars, s.d.

Fig. 3.

Correlation of germline expression changes between P-granule and csr-1-depleted worms. (A) Proportional Venn diagram comparing both set 1 (less stringent) and set 2 (highly stringent) datasets from csr-1 and P-granule RNAi experiments. (B) Correlation plots comparing csr-1 and P-granule RNAi log2 fold changes.

Fig. 4.

Distal expansion of msp-3 expression following P-granule and csr-1 RNAi. (A) Representative images show the left gonad arm in fixed whole worms at L4, young adult and day 3 adult stages. Expansion of msp-3 expression is observed following either P-granule or csr-1 RNAi. (B) msp-3 expansion (red) following P-granule or csr-1 RNAi pushes back or suppresses the expression of LIN-41::GFP, an early marker of oocyte development. Red, msp-3 FISH probe; blue, DAPI/DNA; green, PGL-1::GFP in A and LIN-41::GFP in B. Dashed lines indicate domain of msp-3 expression.

Fig. 5.

Model for P-granule and CSR-1 function in the hermaphrodite germline. (A) In germ cells, CSR-1 and PGL-1/3 repress alg-3/4 and sperm-specific transcription. As PGL-1/3 begins to disassemble in primary spermatocytes, factors like ALG-3/4 and IFE-1 promote the transcription and translation of sperm-specific mRNAs. When spermatogenesis completes in C. elegans hermaphrodites, P-granule and CSR-1 expression promotes oocyte development and maturation. Dotted lines as previously proposed in Amiri et al. (2001); Conine et al. (2013). (B) P granules and CSR-1 repress her-1, fog-3 and sperm-specific transcription. When P-granule or CSR-1 function is impaired, her-1 transcription is increased, promoting fog-3 expression and sperm transcription.