RESUMO
The piRNA pathway is a piRNA-guided retrotransposon silencing system which includes processing of retrotransposon transcripts by PIWI-piRNAs in secondary piRNA biogenesis. Although several proteins participate in the piRNA pathway, the ones crucial for the cleavage of target RNAs by PIWI-piRNAs have not been identified. Here, we show that GTSF1, an essential factor for retrotransposon silencing in male germ cells in mice, associates with both MILI and MIWI2, mouse PIWI proteins that function in prospermatogonia. GTSF1 deficiency leads to a severe defect in the production of secondary piRNAs, which are generated from target RNAs of PIWI-piRNAs. Furthermore, in Gtsf1 mutants, a known target RNA of PIWI-piRNAs is left unsliced at the cleavage site, and the generation of secondary piRNAs from this transcript is defective. Our findings indicate that GTSF1 is a crucial factor for the slicing of target RNAs by PIWI-piRNAs and thus affects secondary piRNA biogenesis in prospermatogonia.
Assuntos
Regulação da Expressão Gênica , Proteínas/metabolismo , RNA Interferente Pequeno/genética , Transcrição Gênica , Células-Tronco Germinativas Adultas/metabolismo , Animais , Núcleo Celular/metabolismo , Amplificação de Genes , Inativação Gênica , Genes de Partícula A Intracisternal , Peptídeos e Proteínas de Sinalização Intracelular , Elementos Nucleotídeos Longos e Dispersos , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas/genética , Interferência de RNA , Proteínas Recombinantes de Fusão , Retroelementos , Testículo/metabolismoRESUMO
The unknown protein family 0224 (UPF0224) includes three members that are expressed in germ-line cells in mice: Gtsf1, Gtsf1l, and BC048502 (Gtsf2). These genes produce proteins with two repeats of the CHHC Zn-finger domain, a predicted RNA-binding motif, in the N terminus. We previously reported that Gtsf1 is essential for spermatogenesis and retrotransposon suppression. In this study, we investigated the expression patterns and functions of Gtsf1l and Gtsf2. Interestingly, Gtsf1l and Gtsf2 were found to be sequentially but not simultaneously expressed in gonocytes and spermatids. Pull-down experiments showed that both GTSF1L and GTSF2 can interact with PIWI-protein complexes. Nevertheless, knocking out Gtsf1, Gtsf2, or both did not cause defects in spermatogenesis or retrotransposon suppression in mice.
