Cytoplasmic Compartmentalization of the Fetal piRNA Pathway in Mice

Abstract

Derepression of transposable elements (TEs) in the course of epigenetic reprogramming of the mouse embryonic germline necessitates the existence of a robust defense that is comprised of PIWI/piRNA pathway and de novo DNA methylation machinery. To gain further insight into biogenesis and function of piRNAs, we studied the intracellular localization of piRNA pathway components and used the combination of genetic, molecular, and cell biological approaches to examine the performance of the piRNA pathway in germ cells of mice lacking Maelstrom (MAEL), an evolutionarily conserved protein implicated in transposon silencing in fruit flies and mice. Here we show that principal components of the fetal piRNA pathway, MILI and MIWI2 proteins, localize to two distinct types of germinal cytoplasmic granules and exhibit differential association with components of the mRNA degradation/translational repression machinery. The first type of granules, pi-bodies, contains the MILI-TDRD1 module of the piRNA pathway and is likely equivalent to the enigmatic “cementing material” first described in electron micrographs of rat gonocytes over 35 years ago. The second type of granules, piP-bodies, harbors the MIWI2-TDRD9-MAEL module of the piRNA pathway and signature components of P-bodies, GW182, DCP1a, DDX6/p54, and XRN1 proteins. piP-bodies are found predominantly in the proximity of pi-bodies and the two frequently share mouse VASA homolog (MVH) protein, an RNA helicase. In Mael-mutant gonocytes, MIWI2, TDRD9, and MVH are lost from piP-bodies, whereas no effects on pi-body composition are observed. Further analysis revealed that MAEL appears to specifically facilitate MIWI2-dependent aspects of the piRNA pathway including biogenesis of secondary piRNAs, de novo DNA methylation, and efficient downregulation of TEs. Cumulatively, our data reveal elaborate cytoplasmic compartmentalization of the fetal piRNA pathway that relies on MAEL function.

Transposable elements in the mammalian germline: a comfortable niche or a deadly trap?

“…This phenotype was originally described for the methylationdeficient Dnmt3L mutant males (Bourc'his and Bestor, 2004), observed again in PIWI mutants (Carmell et al, 2007;Aravin et al, 2007b) and more recently recapitulated in mutations for proteins supporting the piRNA/ PIWI pathway, such as the Tudor domain-containing proteins TDRD1 and TDRD9 (Ollinger et al, 2008;Soper et al, 2008;Ma et al, 2009;Reuter et al, 2009;Shoji et al, 2009). Tudor domain-containing proteins assemble specialized RNA processing platforms in the cytoplasm of germ cells, to which they recruit PIWI proteins through their ability to bind symmetrically dimethylated arginines, a post-translational modification undergone by both MILI and MIWI2 (Aravin et al, 2009;Vagin et al, 2009;Wang et al, 2009;Shoji et al, 2009). As a whole, directed mutagenesis in mice has allowed the identification of nine proteins with a key role in safe guarding the male germline against TEs (Table 1 and Figure 1): Dnmt3L, Dnmt3A, MIWI2, MILI, Maelstrom (MAEL), TDRD1, TDRD9, GASZ and Tex19.1 (Testis expressed 19.1) (Ollinger et al, 2008;Soper et al, 2008;Ma et al, 2009).…”

“…Pi-bodies were recently shown to contain another important player of fetal piRNA biogenesis, the GASZ protein whose biochemical function is unknown (Germ cell protein with Ankyrin repeats, Sterile alpha motifs and Leucine Zipper) . Secondary antisense piRNAs are more prone to associate with MIWI2 proteins, which interact with other specialized Tudor proteins, TDRD9, and localize to distinct compartments called 'piP-bodies' (Aravin et al, 2009;Shoji et al, 2009). Exchange of sense and antisense piRNAs between pi-and piP-bodies intensifies and accelerates the degradation of TE transcripts, which eventually leads to the accumulation of TE-associated piRNAs in prospermatogonia.…”

mRNP granules

“…Thus, mRNA decay blocks could also affect germ granules and neuronal transport granules, with which P-bodies also physically interact. 14,15 Notably, various mRNA degradative enzymes are found in mRNP granules besides P-bodies, such as Xrn1 in stress granules 11 and Drosophila/mouse spermatid nuage, 65 Dcp1/ Dcp2 in various germ granules, [66][67][68] and several Argonaute proteins with endonuclease activity in stress granules, 69 and various germ granules. [70][71][72][73] Whether mRNA degradation occurs in mRNP granules besides P-bodies remains poorly studied.…”

“…However, ribosomes are a component of many neuronal transport, and decay factors are also often found in neuronal and germ granules, 104 and sometimes are even required for their localization. 67 Thus, one assumes that the mRNP state within these granules is carefully regulated. Why transport factors involved in processes that are normally suppressed during localization?…”

Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus

“…They interact with PIWIL2 or PIWIL1 (MIWI) (Kuramochi-Miyagawa et al 2001;Deng and Lin 2002) and have diverse sequences that are mapped in clusters in unannotated regions. PIWIL1 is localized to perinuclear structures called chromatoid bodies (Grivna et al 2006b;Kotaja et al 2006;Aravin et al 2009;Chuma et al 2009), which are the presumed sites of PTGS, analogous to the processing bodies in somatic cells (Liu et al 2005). However, since only 17%-20% of the total pachytene piRNAs corresponds to retrotransposons (Aravin et al 2006;Girard et al 2006;Grivna et al 2006a;Lau et al 2006;Watanabe et al 2006), their role in retrotransposon silencing is less clear.…”