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, 18 (12), 861-7

A C. Elegans Piwi, PRG-1, Regulates 21U-RNAs During Spermatogenesis

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A C. Elegans Piwi, PRG-1, Regulates 21U-RNAs During Spermatogenesis

Guilin Wang et al. Curr Biol.

Abstract

Background: Epigenetic regulation by diverse classes of small RNAs is mediated by the highly conserved Argonaute/Piwi family of proteins. Although Argonautes are broadly expressed, the Piwi subfamily primarily functions in the germ line. Piwi proteins are associated with germline-specific ribonucleoprotein (RNP) granules in Drosophila, zebrafish, and mouse. Depending on the species and on the specific family member, Piwis play important roles in spermatogenesis and/or in maintaining germ cell and stem cell totipotency. Piwis bind to a newly discovered class of small RNAs, called piRNAs. C. elegans contains a large set of Argonaute/Piwi-related proteins, including two closely related to piwi called prg-1 and prg-2. The function of prg-1 and prg-2 and whether piRNAs exist in C. elegans is unknown.

Results: Here, we demonstrate that the Piwi-like protein PRG-1 is localized to P granules in germ cells entering spermatogenesis and is required for successful spermatogenesis. Loss of prg-1 causes a marked reduction in expression of a subset of mRNAs expressed during spermatogenesis, and prg-1 mutant sperm exhibit extensive defects in activation and fertilization. Moreover, prg-1 activity is required for the presence of the small RNAs called 21U-RNAs.

Conclusions: Our data suggest that PRG-1 promotes expression, processing, or stability of 21U-RNAs, which, in turn or in concert with PRG-1, promote proper expression of spermatogenesis transcripts.

Figures

Figure 1
Figure 1. Disruption of prg-1 causes reduction in brood size and temperature-sensitive sterility
A) Schematic of prg-1 and prg-2 genomic structure. Box, exon; connecting line, intron. Functional domains (PAZ, MID and PIWI) of PRG proteins are indicated below. The solid lines above indicate the deletions in prg-1(tm872) and prg-2(tm1094). B) The average number of germ cells per gonad positive for phosphorylated Ser10 of histone H3 at 25°C is graphed. The X-axis indicates genotype; the Y-axis represents mean number of pH3+ cells. N>10 gonads. C) Dissected gonad arms from different genotypes stained with DAPI. Asterisk marks distal end of gonad. D) prg-1 mutants have a temperature sensitive sterile phenotype. Brood size of wild-type or mutant worms was determined at 20°C or 25 °C. The X-axis indicates corresponding genotypes; the Y-axis is the mean value of total number of progeny per animal. Greater than eight animals per trial, average of three trials. Error bars indicate standard deviation.
Figure 2
Figure 2. prg-1(tm872) mutants exhibit defects in spermatogenesis
A) Wild type or prg-1(tm872) L4 hermaphrodites were either left unmated or mated to wild type males at 20°C, and the number of progeny counted. X-axis = genotypes, Y-axis = mean progeny per animal. N ≥16. Error bars indicate standard deviation. B) Wild type or prg-1(tm872) males were mated with prg-1, fem-1 or unc-32 mutant hermaphrodites at 25°C, and the number of progeny counted (cross-progeny only for unc-32). X-axis = genotypes, Y-axis = mean progeny per animal. Greater than eight animals per trial, two or three trials averaged. Error bars indicate standard deviation. C) Young adult hermaphrodites or males were examined by microscopy under DIC. Arrows indicate the first oocyte produced after completing spermatogenesis in hermaphrodites. Triangles indicate spermatids. Scale bar: 10μm. D) Image of pronase-treated spermatids in wild type and prg-1 mutants. Scale bar: 5μm. Arrow: activated spermatids; asterisk: unactivated spermatids. E) Quantification of spermatid activation, N>137 spermatids.
Figure 3
Figure 3. PRG-1 localizes to P granules during spermatogenesis
A) Live animals expressing the PRG-1:GFP transgene were exposed to Hoechst dye, and then gonads directly dissected and mounted for viewing. DIC and fluorescent images were collected. Scale bar: 10μm. B) The pachytene region of L4 and adult gonads from PRG-1::GFP transgenic animals are shown. Green, PRG-1::GFP; blue, Hoechst; scale bar, 10μm. C) PRG-1:GFP intensity was measured from equivalent exposures of pachytene germ cells from 10 animals each at L4 and adult stages. Error bar indicates standard deviation. * = Student’s t-test, P<0.01 D) L4 hermaphrodite gonads were stained with anti-GFP (green) and anti-PGL-1 (red). Upper panels: 1000X magnification; scale bar, 5 μm; lower panels: 400X magnification.
Figure 4
Figure 4. prg-1- regulated genes exhibit the same chromosome bias as genes expressed during spermatogenesis
A total of 1300 genes known to be expressed during spermatogenesis and the 529 genes downregulated by prg-1 were mapped to their chromosomal locations and the numbers compared to the expected number given the representation of each chromosome on the microarray. * = p<0.01 (Chi square).
Figure 5
Figure 5. prg-1 mutants fail to express 21U RNAs
A) Temporal expression of 21ur-1 in wild-type and prg-1(tm872) worms at 20°C and 25°C. B) 21ur-1 RNA expression in mutants with defects in germline development: glp-1 has almost no germ cells; fem-1 has oocytes only, and fem-3 has sperm only. Numbers indicate the relative intensity ratio of 21ur-1 to let-7, after comparison to wild type, which was set to one. Ethidium bromide staining of tRNA shows similar loading levels.

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