Mouse GTSF 1 is an essential factor for secondary pi RNA biogenesis
2018; Springer Nature; Volume: 19; Issue: 4 Linguagem: Inglês
10.15252/embr.201642054
ISSN1469-3178
AutoresTakuji Yoshimura, Toshiaki Watanabe, Satomi Kuramochi‐Miyagawa, Noriaki Takemoto, Yusuke Shiromoto, Akihiko Kudo, Masami Kanai‐Azuma, Fumi Tashiro, Satsuki Miyazaki, Ami Katanaya, Shinichiro Chuma, Jun‐ichi Miyazaki,
Tópico(s)Animal Genetics and Reproduction
ResumoArticle7 February 2018free access Transparent process Mouse GTSF1 is an essential factor for secondary piRNA biogenesis Takuji Yoshimura orcid.org/0000-0002-7856-0679 Laboratory of Reproductive Engineering, The Institute of Experimental Animal Sciences, Osaka University Medical School, Suita, Osaka, Japan Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Toshiaki Watanabe Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Central Institute for Experimental Animals, Kawasaki-ku, Kawasaki, Kanagawa, Japan Search for more papers by this author Satomi Kuramochi-Miyagawa orcid.org/0000-0003-4396-5868 Department of Pathology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Noriaki Takemoto Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Yusuke Shiromoto Department of Pathology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Akihiko Kudo Department of Anatomy, Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo, Japan Search for more papers by this author Masami Kanai-Azuma Center for Experimental Animal, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Fumi Tashiro Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Satsuki Miyazaki Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Ami Katanaya Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Search for more papers by this author Shinichiro Chuma Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Search for more papers by this author Jun-ichi Miyazaki Corresponding Author [email protected] orcid.org/0000-0003-2475-589X Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Takuji Yoshimura orcid.org/0000-0002-7856-0679 Laboratory of Reproductive Engineering, The Institute of Experimental Animal Sciences, Osaka University Medical School, Suita, Osaka, Japan Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Toshiaki Watanabe Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Central Institute for Experimental Animals, Kawasaki-ku, Kawasaki, Kanagawa, Japan Search for more papers by this author Satomi Kuramochi-Miyagawa orcid.org/0000-0003-4396-5868 Department of Pathology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Noriaki Takemoto Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Yusuke Shiromoto Department of Pathology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Akihiko Kudo Department of Anatomy, Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo, Japan Search for more papers by this author Masami Kanai-Azuma Center for Experimental Animal, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Fumi Tashiro Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Satsuki Miyazaki Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Ami Katanaya Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Search for more papers by this author Shinichiro Chuma Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Search for more papers by this author Jun-ichi Miyazaki Corresponding Author [email protected] orcid.org/0000-0003-2475-589X Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Search for more papers by this author Author Information Takuji Yoshimura1,2, Toshiaki Watanabe3,4,5, Satomi Kuramochi-Miyagawa6, Noriaki Takemoto2, Yusuke Shiromoto6, Akihiko Kudo7, Masami Kanai-Azuma8, Fumi Tashiro2, Satsuki Miyazaki2, Ami Katanaya9, Shinichiro Chuma9 and Jun-ichi Miyazaki *,2 1Laboratory of Reproductive Engineering, The Institute of Experimental Animal Sciences, Osaka University Medical School, Suita, Osaka, Japan 2Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Suita, Osaka, Japan 3Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA 4Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA 5Central Institute for Experimental Animals, Kawasaki-ku, Kawasaki, Kanagawa, Japan 6Department of Pathology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Suita, Osaka, Japan 7Department of Anatomy, Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo, Japan 8Center for Experimental Animal, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan 9Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan *Corresponding author. Tel: +81 6 6105 5934; E-mail: [email protected] EMBO Rep (2018)19:e42054https://doi.org/10.15252/embr.201642054 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis Mouse GTSF1 is essential for MILI-directed secondary piRNA biogenesis in prospermatogonia and associates with the MIWI2 complex probably to transcriptionally silence its targets. Mouse GTSF1 associates with MILI, MIWI, and MIWI2 complexes. Mouse GTSF1 deficiency leads to loss of MIWI2-bound piRNAs and to a severe defect in secondary piRNA biogenesis, whereas it has no impact on primary piRNA biogenesis. 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. Introduction Retrotransposons are mobile genetic elements that autonomously replicate and insert into the host genome upon their derepression. Retrotransposon activity is potentially threatening for transgenerational genome stability in germ cells and their precursors 1. Thus, the germline has evolved mechanisms to suppress retrotransposons, including the PIWI-interacting RNA (piRNA) pathway, a small RNA-guided silencing system 23. A number of piRNA pathway proteins essential for retrotransposon suppression have been identified 4. In mice, PIWI genes encode the core proteins MILI and MIWI2, which play central roles in the piRNA pathway in prospermatogonia, where paternal imprinting and de novo DNA methylation take place 56. piRNA biogenesis largely consists of two pathways: primary and secondary piRNA biogenesis pathways. Primary piRNAs are derived from transcripts including transposons, genic mRNAs, noncoding RNAs, and piRNA clusters, through a processing mechanism that involves the helicase MOV10L1, and characteristically have a uridine at the 5′ end (1U) 7. MILI binds directly to primary piRNAs and processes target RNAs according to the guide sequence of primary piRNAs to generate secondary piRNAs, which often have adenine at 10th nucleotide (10A) because of the 1U in primary piRNAs 8. The generated secondary piRNAs can process target RNAs according to the guide sequence of the secondary piRNAs to reproduce piRNAs having the same sequence as primary piRNAs. This system of repeated piRNA production is called the ping-pong cycle. The slicer activity of MILI is essential for secondary piRNA biogenesis and the ping-pong cycle 9. Thus, posttranscriptional silencing of retrotransposons is mediated in part by transcript cleavage and processing 10. Binding of the secondary piRNA to MIWI2 results in the formation of a piRNA-bound MIWI2 complex that is thought to recognize transposon targets in the host genome and to recruit components, including the catalytically inactive DNA methyltransferase DNMT3L, to silence them by DNA methylation 8. The putative MIWI2 catalytic domain for slicer activity is not required for secondary piRNA biogenesis and transposon suppression 9. Thus, retrotransposons are also silenced pretranscriptionally by epigenetic regulation. Several piRNA pathway components reside in cytoplasmic granules termed “nuages”, which are germline-specific organelles that are classified into two distinct types 11. One type consists of MILI-containing granules, which are intermitochondrial cement-like granules (or pi-bodies) that co-localize with the Tudor protein TDRD1, a direct binding partner of MILI 121314. The second type consists of MIWI2-containing granules, which are processing bodies (or piP-bodies) that are larger and less abundant than pi-bodies, and co-localize with TDRD9, a direct binding partner of MIWI2 15. Several Tudor proteins are known to act as adaptor molecules through their Tudor domains that bind to effector proteins such as PIWI proteins, and are involved in the piRNA pathway 16. We previously reported that the mouse Gtsf1 gene, which encodes gametocyte-specific factor 1 (GTSF1), is expressed preferentially in germ cells and that Gtsf1-null male mice are sterile 1718. Detailed analyses of these mice revealed that Gtsf1 is essential for meiosis progression beyond early prophase I during spermatogenesis and that its loss results in elevated expression of long interspersed nucleotide element-1 (LINE-1) and intracisternal A-particle (IAP) retrotransposons, accompanied by demethylation of their promoter regions 18. Therefore, mouse Gtsf1 was identified as a gene involved in retrotransposon suppression in male germ cells. Previously, three groups demonstrated that the Drosophila Gtsf1 protein (also known as DmGTSF1 or Asterix) interacts with Piwi complex and is an essential component of the piRNA-guided transcriptional silencing complex in ovarian germline and somatic cells 192021. However, the role of mouse GTSF1 in the piRNA pathway is currently unclear. In this study, we demonstrated that GTSF1 is a component of both MILI and MIWI2 complexes, and lack of GTSF1 in mouse prospermatogonia leads to derepressed LINE-1 and IAP expression, aberrant localization of several major piRNA pathway components, and defective secondary piRNA biogenesis. Further, we observed that a noncoding RNA known to be targeted by piRNAs remained unsliced in Gtsf1−/− prospermatogonia. These data indicate that GTSF1 has crucial role(s) in secondary piRNA biogenesis by regulating piRNA-mediated cleavage of target RNAs. Results Loss of Gtsf1 leads to derepression of LINE-1 and IAP in prospermatogonia We previously reported that the loss of Gtsf1 leads to the derepression of LINE-1 and IAP in the testes at postnatal day (P) 14, which precedes the time point at which germ cell defects can be histologically detected 18. Here, we examined LINE-1 and IAP expression in the earlier developmental stages, embryonic day (E) 17.5, P0, P4, and P8, by immunofluorescence analysis of L1 ORF1p, an active LINE-1 element protein product 22, and IAP GAG, an IAP protein product 23. Increased LINE-1 and IAP expression was detected as early as E17.5 in Gtsf1−/− testes as compared to Gtsf1+/− testes (Figs 1A and B, and EV1A). These results indicated that GTSF1 is essential for repressing LINE-1 and IAP expression during the development from prospermatogonia to spermatogonia. Figure 1. Loss of piP-body component GTSF1 affects localization of other piP-body components, whereas loss of pi-body component MILI affects GTSF1 localization A, B. Loss of Gtsf1 elevates retrotransposon expression in prospermatogonia. Immunostaining of Gtsf1+/− and Gtsf1−/− E17.5 testes with anti-L1 ORF1p (A, green) and anti-IAP GAG (B, green) antibodies. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) (red). Scale bar, 10 μm. C–G. GTSF1 localizes to piP-bodies and nuclei in prospermatogonia. Schematic representation of the timing of expression of Gtsf1 and Piwi family genes in mouse (C). Immunofluorescence analysis of Gtsf1+/− and Gtsf1−/− E17.5 prospermatogonia using an anti-GTSF1 antibody (green) and DAPI (red) for DNA staining (D). GTSF1 localizes to cytoplasmic granules and nuclei. Double staining of GTSF1 with TDRD9 (E), MIWI2 (F), and MILI (G). DNA was stained with DAPI (blue). In (G), lower panels are magnified views of double staining of GTSF1 with MILI in a GTSF1 focus in the upper panel. GTSF1 foci consistently co-stained with TDRD9 and MIWI2 foci (E, F) and overlapped with MILI foci (G). Scale bar, 10 μm. H–M. Loss of Gtsf1 results in abnormal localization of piP-body components. Immunostaining of Gtsf1+/− and Gtsf1−/− E17.5 testes with antibodies to MAEL (H), TDRD9 (I), MIWI2 (J), MILI (K), MVH (L), and TDRD1 (M). DNA was stained with DAPI (red). MIWI2, MAEL, and TDRD9 lost their localization to piP-bodies in the Gtsf1−/− prospermatogonia, whereas the localization of MVH, TDRD1, and MILI to pi-body was unaffected. Nuclear localization of MIWI2 was lost in Gtsf1−/− prospermatogonia. Scale bar, 10 μm. N–P. Loss of Mili, but not of Miwi2 or Tdrd9, abrogates GTSF1 localization to piP-bodies. Anti-GTSF1 antibody immunostaining (green) of (N) Mili+/− and Mili−/− E17.5 testes, (O) Miwi2+/− and Miwi2−/− E17.5 testes, and (P) Tdrd9+/− and Tdrd9−/− E17.5 testes. DNA was stained with DAPI (red). Scale bar, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Immunofluorescence and electron microscopic analyses of Gtsf1-deficient testes (related to Fig 1) Elevated expression of LINE-1 and IAP in early postnatal periods caused by Gtsf1 deficiency. Immunostaining of Gtsf1+/− and Gtsf1−/− testes at postnatal days 0, 4, and 8 with antibody to Line-1 ORF1 protein (left panel, green) and IAP GAG protein (right panel, green). DNA was stained with DAPI (red). Scale bar, 10 μm. Expression of GTSF1 in early postnatal periods. Immunostaining of Gtsf1+/− and Gtsf1−/− testes at postnatal days 0, 4, and 8 with antibody to GTSF1 (green). DNA was stained with DAPI (red). Scale bar, 10 μm. Histological analysis of Gtsf1-deficiency phenotypes. Immunostaining of Gtsf1+/− and Gtsf1−/− E17.5 testes with anti-DNMT3A2 (upper panel, green) and anti-DNMT3L (lower panel, green) antibodies. DNA was stained with DAPI (red). Localization of DNMT3L and DNMT3A/3A2 was unaffected in the prenatal Gtsf1−/− testes. Scale bar, 10 μm. Electron microscopy of Gtsf1+/− (upper panel) and Gtsf1−/− (lower panel) E17.5 prospermatogonia. Right panels (scale bar, 1 μm) are magnified views of the boxed region in the left panels (scale bar, 5 μm). Arrows indicate intermitochondrial cement. Download figure Download PowerPoint Mouse GTSF1 localizes to piP-bodies and nuclei in prospermatogonia Elevated LINE-1 and IAP expression in the prospermatogonia of prenatal gonads has also been observed in mice harboring null mutations in the genes encoding piRNA pathway components 9242526272829. In prenatal prospermatogonia at E17.5, these components localize to two types of granules, pi-bodies and piP-bodies. In Drosophila, DmGTSF1 localizes specifically to the nuclei of ovarian germline and somatic support cells, not to cytoplasmic granules 2021. We previously showed that mouse GTSF1 localizes to cytoplasmic granules in the spermatocytes and spermatids of the adult testes and in the prospermatogonia of E17.5 testes, by immunostaining with anti-GTSF1 antibody (Fig 1C) 1718. To further clarify the localization of GTSF1, we here conducted immunofluorescence analysis using an optimized staining method. We found that GTSF1 localized not only to the cytoplasmic granules but also to the nuclei in the prospermatogonia of E17.5 testes (Fig 1D). At later developmental time points, P0, P4, and P8, cytoplasmic staining was detected although the prominent cytoplasmic GTSF1 foci were gradually lost over time (Fig EV1B). By double immunostaining, we found that the GTSF1 foci completely co-localized with TDRD9 (Fig 1E) and MIWI2 (Fig 1F) foci, both of which are piP-body components 1115. In contrast, while all GTSF1 foci co-localized or overlapped with MILI foci, GTSF1 foci appeared to be less abundant than MILI foci (Fig 1G). These observations are consistent with the report that MIWI2 foci co-stain with or are in close proximity to MILI foci 8. Taken together, these findings suggested that GTSF1 is a component of piP-bodies in prospermatogonia. piRNA pathway components of piP-bodies are mislocalized in Gtsf1-deficient prospermatogonia The impact of Gtsf1 disruption on the localization of the piP-body components, MIWI2, TDRD9, and MAEL, was examined by immunofluorescence analysis of E17.5 Gtsf1+/− and Gtsf1−/− testes. MAEL is an evolutionarily conserved protein involved in the piRNA pathway 1129. MAEL-positive granules were clearly observed as large foci in Gtsf1+/− prospermatogonia, while granules were only weekly stained in Gtsf1−/− prospermatogonia (Fig 1H). TDRD9-positive granules were completely lost in Gtsf1−/− prospermatogonia. However, the uniform nuclear staining of TDRD9 was unchanged (Fig 1I), indicating that GTSF1 is not required for the nuclear localization of TDRD9. Similarly, the nuclear localization of DNMT3L and DNMT3A2, which are essential for de novo DNA methylation of retrotransposons, appeared to be unaffected by Gtsf1 disruption (Fig EV1C). In contrast, the nuclear localization of MIWI2 disappeared in Gtsf1−/− prospermatogonia, and MIWI2 staining was found only in the cytoplasm, without prominent foci (Fig 1J). Taken together, these findings indicated that GTSF1 deficiency alters the granular localization of MAEL and TDRD9 and severely affects the localization of MIWI2 in prospermatogonia, implying that GTSF1 has a strong influence on the piRNA pathway-associated components of piP-bodies. Next, we examined the effects of Gtsf1 disruption on the localization of the pi-body components MILI, MVH, and TDRD1. MVH is an evolutionarily conserved helicase that plays an essential role in the piRNA pathway 26. The MILI-, MVH-, and TDRD1-stained granules exhibited similar staining patterns between Gtsf1+/− and Gtsf1−/− (Fig 1K–M), but the MILI-stained granules appeared to be more intensely stained in Gtsf1−/− than in Gtsf1+/− prospermatogonia (Fig 1K). The fine structure of pi-bodies was analyzed by electron microscopy, which showed the presence of electron-dense intermitochondrial cement in both Gtsf1+/− and Gtsf1−/− prospermatogonia (Fig EV1D). These results demonstrated that GTSF1 deficiency appears to have limited impact on the structure of pi-bodies and the localization of their components. To further investigate the functional relationship between GTSF1 and the other piRNA pathway components, we examined the subcellular localization of GTSF1 in MILI-, MIWI2-, and TDRD9-deficient prospermatogonia. Notably, cytoplasmic granular localization of GTSF1 was completely lost in prospermatogonia of Mili−/− (Fig 1N), but was unaffected in those of Miwi2−/− (Fig 1O) and Tdrd9−/− (Fig 1P) mice. These results indicated that MILI is crucial for GTSF1 localization into piP-bodies, and therefore may impact the molecular function of GTSF1 in prospermatogonia. GTSF1 associates with both MILI and MIWI2 complexes The above findings that GTSF1 localizes to the piP-bodies (Fig 1E–G) and that loss of GTSF1 leads to mislocalization of the piP-body components (Fig 1H–J) suggested that GTSF1 interacts with piRNA pathway-associated granule components. To identify the protein complexes from prenatal or adult testes that can associate with GTSF1, pull-down experiments were carried out using recombinant full-length GTSF1-glutathione S-transferase (GST)-fusion proteins (Fig 2A). Complexes from E17.5 testes containing MILI, MIWI2, TDRD1, or TDRD9 interacted with full-length GTSF1 (Fig 2B lane FL). Similarly, complexes from adult testes containing MILI, MIWI, TDRD1, or TDRD9 interacted with GTSF1 (Fig EV2A lane FL). To identify the region(s) in GTSF1 essential for these interactions, the following truncated GTSF1 derivatives were produced, fused with GST, and used in GST pull-down experiments (Fig 2A): (i) ΔC, lacking the C-terminal region; (ii) ZnF, containing only the N-terminal Zn-finger region; and (iii) CR, containing only the central region. The results showed that the C-terminal domain of GTSF1 was not required for interactions with either PIWI or Tudor-containing complexes (Figs 2B and EV2A) and that the central region of GTSF1 was sufficient for interactions with MILI- and MIWI2-containing complexes (Fig 2B). It is noteworthy that RNase A treatment considerably reduced the interaction of GTSF1 with MIWI2 (Fig 2B), but not with MILI or MIWI complexes (Figs 2B and EV2A), suggesting that RNA in the MIWI2 complex is required for the binding of GTSF1. Figure 2. GTSF1 interacts with PIWI protein complexes Schematic illustration of the GTSF1 protein with two N-terminal CHHC-type Zn fingers, and the following deletion fragments used to generate GST-fusion proteins for pull-down experiments: FL (full length), ΔC (C-terminal deletion), ZnF (Zn-finger region), and CR (central region). In addition, CR fragments containing one or two alanine substitutions at W98, W107, or W112 (red stars) were used to generate GST-fusion proteins for pull-down analysis. GST pull-down analysis of the interaction of GTSF1 with MILI, MIWI2, TDRD1, or TDRD9. GST-fusion proteins bound to glutathione sepharose were incubated with E17.5 testis lysates. In some experiments, testis lysates were pretreated with RNase A prior to the incubation with GST-fusion proteins. The proteins bound to GST-fusion proteins were analyzed by SDS–PAGE followed by Western blotting with antibodies to MILI, MIWI2, TDRD1, and TDRD9 (upper panels). Coomassie brilliant blue (CBB) staining shows the amount of the GST-fusion proteins in each of the reactions (lower panels). GST pull-down analysis of the interaction of mutated CR proteins with MILI or MIWI2. GST-fusion proteins bound to glutathione sepharose were incubated with E17.5 testis lysates. The proteins bound to GST-fusion proteins were analyzed by SDS–PAGE followed by Western blotting with antibodies to MILI and MIWI2 (upper panels). CBB staining shows the amount of the GST-fusion proteins in each of the reactions (lower panels). Immunoprecipitation analysis of the interaction between GTSF1 and TDRD9. FLAG-tagged TDRD9 and Myc-tagged GTSF1 were expressed in HEK293 cells. Cell lysates were immunoprecipitated with an anti-Myc-tag antibody, followed by SDS–PAGE and Western blotting analysis using an anti-TDRD9 or anti-GTSF1 antibody. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Analysis of GTSF1-associated proteins (related to Fig 2) GST pull-down analysis of the interaction of GTSF1 with MILI, MIWI, TDRD1, or TDRD9. The GST-fusion proteins bound to glutathione sepharose were incubated with adult testis lysates. In some experiments, testis lysates were pretreated with RNase A prior to incubation with GST-fusion proteins. The proteins bound to GST-fusion proteins were analyzed by SDS–PAGE followed by Western blotting with antibodies to MILI, MIWI, TDRD1, and TDRD9 (upper panels). CBB staining shows the amount of the GST-fusion proteins in each of the reactions (lower panels). Schematic illustration of mouse GTSF1 protein. Amino acid sequences in the central regions of five species, including mouse, are shown. Black-and-white inverted characters represent conserved aromatic amino acid residues in the central regions among the species, which were used for alanine substitutions in pull-down analysis. These aromatic amino acid residues are surrounded by several negatively charged amino acid residues (in blue font). GST pull-down analysis of the interaction of mutated CR proteins with MILI or MIWI complexes. The GST-fusion proteins bound to glutathione sepharose were incubated with adult testis lysates. The proteins bound to GST-fusion proteins were analyzed by SDS–PAGE followed by Western blotting with antibodies to MILI and MIWI (upper panels). CBB staining shows the amount of the GST-fusion proteins in each of the reactions (lower panels). Immunoprecipitation analysis of the binding of GTSF1 to several piRNA pathway components. Myc-tagged GTSF1 was co-expressed with FLAG-tagged MAEL, MILI, MIWI, MIWI2, MVH, or His-tagged TDRD1 in HEK293 cells. Lysates of the transfected cells were immunoprecipitated with an anti-Myc antibody and separated by SDS–PAGE, followed by Western blotting using the indicated antibodies. Download figure Download PowerPoint DmGTSF1 interacts with the Piwi complex via its central region, which includes the aromatic residues, tryptophan (W) 89 and tyrosine (Y) 98, which are crucial for interaction with the Piwi complex 20, surrounded by negatively charged amino acids. To identify the residues in mouse GTSF1 that potentially mediate interactions with the PIWI proteins, we searched its amino acid sequence for aromatic residues surrounded by negatively charged amino acids and found three W residues in the CR (Figs 2A and EV2B). We investigated their involvement in interactions with the PIWI complex by generating CR-GST-fusion proteins in which either one or two residues were substituted with alanine and using them in pull-down experiments. The W107A mutation most efficiently abrogated the interaction with the PIWI proteins (Figs 2C and EV2C), suggesting that W107 may be directly or indirectly involved in mediating interactions between GTSF1 and PIWI in mice. To confirm GTSF1 binding to PIWI complexes, we used liquid chromatography–mass spectrometry (LC-MS) to evaluate anti-GTSF1 antibody immunoprecipitates from adult mouse testes. The analysis showed that various piRNA pathway-related proteins, including PIWI proteins, were recovered in the immunoprecipitates from Gtsf1+/− testes, but not from Gtsf1−/− testes or when using normal IgG (Table EV1). Thus, GTSF1 binds to and is a component of PIWI complexes in vivo. Direct interactions between PIWI and Tudor family proteins have been intensely examined 121314271530. On the other hand, direct interaction between DmGTSF1 and Piwi in ovarian somatic cells has been suggested 2021. Here, we examined the direct binding of mouse GTSF1 to other piRNA pathway components by expressing them as tagged proteins in HEK293 or BMT10 cells and then examining their interactions by immunoprecipitation followed by Western blotting analysis. Recombinant GTSF1 bound only to TDRD9 (Fig 2D). We could not detect binding of GTSF1 to MAEL, MILI, MIWI, MIWI2, MVH, or TDRD1 (Fig EV2D). Therefore, mouse GTSF1 may bind to PIWI proteins with the help of or via other factors. Lack of GTSF1 results in loss of MIWI2-bound piRNAs To investigate the impact of GTSF1 deficiency on piRNA biogenesis, we deep-sequenced total small RNAs from Gtsf1+/− and Gtsf1−/− E17.5 testes. We mapped the reads to the mouse reference genome, and their origins were annotated (Dataset EV1; Fig EV3A). The read-length distribution profiles revealed two discernible groups of small RNAs that represented miRNAs [19–22 nucleotides (nt)] and piRNAs (24–30 nt) in the libraries from both Gtsf1+/− and Gtsf1−/− E17.5 testes (Fig EV3B). MILI- and MIWI2-bound piRNAs have different size distributions: from 23 to 30 nt, with a peak at 26–27 nt, and from 24 to 31 nt, with a peak at 28 nt, respectively (Fig 3A) 8. Comparison of the small RNA size profiles of the libraries suggested that the size profile of MIWI2-bound piRNAs appeared to be ablated in the Gtsf1−/− libraries although that of MILI-bound piRNAs appeared to be intact (Fig 3B). This was obvious in small RNAs derived from LINEs and long terminal repeats (LTRs) (Fig 3C). Furthermore, in Gtsf1−/− E17.5 testes, nuclear MIWI2 staining was lost (Fig 1J). This phenotype is commonly observed in several piRNA pathway mutant mice, in which loading of MIWI2 with piRN
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