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.

Figure 1. Localization of MAEL, MIWI2, and MILI to cytoplasmic granules in gonocytes.

(A–C’) Sections of E16.5 (A, A’), E18.5 (B, B’) and P2 (C, C’) testes stained with anti-MAEL antibody (green) and DAPI (blue). (A’–C’) anti-MAEL signal in black/white to emphasize nuclear signal. (D) Localization of MIWI2 and MAEL in a E18.5 gonocyte. MAEL was detected by a directly labeled antibody, resulting in a diminished detection level. (E) Localization of MILI and MAEL in a E18.5 gonocyte. MAEL was detected with a directly labeled antibody, resulting in a diminished detection level. (F–G) Localization of MAEL (F) and MILI (G) in E18.5 testes by immuno-electron microscopy. Localization of MAEL shows it to be a component of electron dense perinuclear granules. Immuno-gold labeling for MILI identified the granules observed by IF to be intermitochondrial cement (ICM). The inset shows a blow-up of the region indicated in the red box.

Figure 2. Localization of germ cell specific proteins to granules.

(A–E) Localization of germ cell specific proteins MVH (A, B), TDRD1 (C, D) and TDRD9 (E) combined with MAEL (A, C, E) or MILI (B, D). DNA is labeled with DAPI (blue). (A) MAEL and MVH co-localize in larger granules. MVH additionally localizes to smaller granules. (B) Double labeling with MILI and MVH reveals the latter to be a component of IMC. (C) TDRD1 is a known component of IMC. It localizes to numerous smaller granules that are frequently adjacent to MAEL granules. (D) MILI and TDRD1 co-localize to IMC. (E) TDRD9 co-localizes with MAEL in granules.

Figure 3. The MIWI2/MAEL granule is a modified P-body.

(A–D) Localization of P-bodies in gonocytes. Cross section seminiferous tubule from wild-type testis (E18.5) were probed with antibodies against XRN1 (B), GW182 (C) and DCP1a (D). (E–H) Co-localization of MAEL and P-body components DCP1a (E), DDX6 (F), XRN1 (G), and GW182 (H) in piP-bodies.

Figure 4. Altered structure of piP-bodies in the absence of MAEL.

(A–B’) EM images of piP-bodies in E18.5 wild-type (A, A’) and Mael-mutant gonocytes (B, B’). Magnified regions (A’, B’) are indicated in red in (A,B). Mael-deficient gonocytes exhibit change in overall appearance of the MIWI2/MAEL granule (indicated with black arrow, compare (A’, B’)). Association with mitochondria (indicated with asterisks in (D’, E’)) was not affected. (C–E’) The effect of Mael deficiency on (C–C’) MIWI2, (D–D’) TDRD9 and (E–E’) MVH localization in gonocytes. In wild-type testes, MIWI2 localizes to cytoplasmic granules and becomes nuclear at E16.5 (C, left panel). Later (E18.5, middle panel and P2, right panel), MIWI2 localization in the nucleus becomes more pronounced. (C’) In absence of MAEL, MIWI2 does not localize to cytoplasmic granules and nuclear localization is delayed (E16.5, left panel, E18.5, middle panel and P2 right panel). (D–D’) In wild-type gonocytes TDRD9 co-localizes with GW182 in piP bodies. No TDRD9 accumulations are observed in the Mael mutant. (E) In wild-type gonocytes, MVH and DCP1a frequently co-localize in large granules. (E’) In absence of MAEL this association is lost. (F–H) MAEL localization in wild-type (F), Miwi2-deficient (G), and Mili-deficient (H) testes. In Miwi2-mutant animals, MAEL is still recruited to piP-bodies but cytoplasmic levels are significantly higher than in wild-type gonocytes. MAEL localization in Mili-mutant animals is largely disrupted. Virtually no MAEL granules are observed and no nuclear localization is detected.

Figure 5. The effect of Mael deficiency on piRNA expression in gonocytes.

(A) Annotation of small RNAs cloned from testes of wild-type and Mael-mutant animals. The right panel shows the ratio of LINE and LTR piRNA to miRNA. (B) The size range of piRNAs in the mael mutant. Total cellular piRNA populations are composed of two complexes, MILI, with average piRNA length 26 nt. and MIWI2 with average piRNA length 28 nt. The length profile reflects the ratio of both complexes in the cell. In wild-type animals the ratio of does not significantly change between E16.5 and P2. MIWI2-associated piRNAs (28 nt) corresponding to both LINE and LTR retrotransposons are significantly reduced in Mael-mutant animals. (C) The ratio of sense to antisense piRNAs for L1 and IAP retrotransposons. The amount of LINE L1 antisense piRNA is reduced in Mael-deficient animals at P2. In contrast, amount of LTR IAP antisense piRNA remains stable. (D) The ratio of secondary to primary piRNAs for L1 and IAP retrotransposons. The amount of secondary piRNAs that correspond to LINE L1 and LTR IAP is reduced in Mael-deficient animals at P2.

Figure 6. Release of post-transcriptional silencing of L1 elements in Mael-mutant gonocytes.

(A–F’) Localization of L1 encoded ORF1p in testes of wild-type (A–F) and Mael-mutant (A’–F’) animals from E14.5 to P10. Overall levels in wild-type animals decreased after E16.5 whereas in Mael-mutant testis ORF1p levels increased at E14.5 onwards. From P2 on signal for ORF1p gradually decreased.