PIWIL3 forms a complex with TDRKH in mammalian oocytes

Abstract

18PIWIs are crucial guardians of genome integrity, particularly in germ cells. While mammalian 19 PIWIs have been primarily studied in mouse and rat, a homologue for the human PIWIL3 gene 20 is absent in the Muridae family, and hence the unique function of PIWIL3 in germ cells cannot 21 be effectively modeled by mouse knockouts. Herein, we investigated the expression, 22 distribution and interaction of PIWIL3 in bovine oocytes. We localized PIWIL3 to mitochondria, 23 and demonstrated that PIWIL3 expression is stringently controlled both spatially and 24 temporally before and after fertilization. Moreover, we identified PIWIL3 in a mitochondrial-25 recruited three-membered complex with TDRKH and PNLDC1, and demonstrated by 26 mutagenesis that PIWIL3 N-terminal arginine modifications are required for complex 27 assembly. Finally we sequenced the piRNAs bound to PIWIL3-TDRKH-PNLDC1 and report here 28 that about 50% of these piRNAs map to transposable elements, recapitulating the important 29 role of PIWIL3 in maintaining genome integrity in mammalian oocytes. 30 65 5 Results 66 PIWIL3 is expressed in the cytoplasm during oocyte maturation and early embryo 67 development 68 Since the subcellular localization of a protein often dictates the mode of action and 69 accessibility to substrates and cofactors, we sought to trace the intracellular distribution of 70 PIWIL3 over developmental stages, to further elucidate the functional role of PIWIL3. While 71 the intracellular distributions of PIWIL1 , PIWIL2 and PIWIL4 have been elucidated in mouse 72 testis 10,15 and human fetal oocytes 14 , the subcellular compartment(s) in which PIWIL3 exerts 73 its function remains uninvestigated to date. Herein, we follow the distribution and expression 74 pattern of PIWIL3 in bovine oocytes and preimplantation embryos, by transiently expressing 75 EGFP-PIWIL3 fusion proteins. 76 We generated EGFP-PIWIL3 fusion constructs (both N-and C-terminal tagging), performed in 77 vitro transcription and directly injected EGFP-PIWIL3 mRNA into bovine oocytes at the 78 germinal vesicle (GV) stage and 2 pro-nuclei (2PN) stage zygotes after in vitro fertilization. 79 Zygotes were cultured further in vitro for up to 8 days (blastocyst stage), during which 80 embryos at various developmental stages post fertilization were harvested. 81 First we confirmed that the entire constructs were translated in the oocytes. Microinjection 82 of the EGFP-PIWIL3 and PIWIL3-EGFP fusion construct into oocytes resulted in proteins of 130 83 kDa, corresponding to the total length of EGFP (26 kDa) and PIWIL3 (100 kDa) (Figure 1a).84Next, we followed the localization of EGFP-PIWIL3 in bovine oocytes. Starting from the GV 85 stage, EGFP-PIWIL3 was localized largely to the oocyte cytoplasm in a punctate pattern but 86 was excluded from the nucleus, whereas the EGFP control signal was distributed evenly 87 6 throughout the cell. The expression of PIWIL3 remained cytoplasmic throughout 88 preimplantation development but strongly reduced at the blastocyst stage (F...

The birth of piRNAs: how mammalian piRNAs are produced, originated, and evolved

“…Nonetheless, piRNA pathways in the ovaries of humans, bovines, hamster, and macaques do show distinct differences from those in testes with regards to piRNA abundance, piRNA size, modification, piRNA-associated PIWI proteins, and the genomic origins of piRNA species (Rosenkranz et al 2015;Ishino et al 2021;Hasuwa et al 2021;Zhang et al 2021;Loubalova et al 2021;Yang et al 2019;Tan et al 2020), arguing that piRNA pathways are influenced by the sex of the cell lineage. For instance, in hamsters, compared to testis piRNAs, oocyte piRNAs come from a distinct set of intergenic genomic loci whose transcription are not driven by A-MYB.…”

“…However, the activation of siRNAs in oocytes is rodent specific and not found in bovines nor humans, suggesting that this piRNA-independent defense mechanism is not present in oocytes of other mammalian species (Flemr et al 2013 ; Rosenkranz et al 2015 ). Moreover, compared to most other mammals with 4 PIWI genes, rodents lack Piwil3 , which has been shown to be specifically expressed in oocytes in hamsters, bovines, and humans (Yang et al 2019 ; Tan et al 2020 ; Ishino et al 2021 ), suggesting that the Piwil3-piRNA pathway is specifically missing in the rodent lineage. To test whether the sexual dimorphic requirement for a piRNA pathway is specific for the rodent lineage, three independent groups have disrupted the piRNA pathways in hamsters and revealed that piRNA pathways are indeed required for maintaining germline genome integrity in both sexes (Zhang et al 2021 ; Hasuwa et al 2021 ; Loubalova et al 2021 ).…”