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HSP90α plays an important role in piRNA biogenesis and retrotransposon repression in mouse.

Ichiyanagi T, Ichiyanagi K, Ogawa A, Kuramochi-Miyagawa S, Nakano T, Chuma S, Sasaki H, Udono H - Nucleic Acids Res. (2014)

Bottom Line: HSP90, found in all kingdoms of life, is a major chaperone protein regulating many client proteins.Whereas the mutation in Fkbp6 encoding a co-chaperone reduced piRNAs of 28-32 nucleotides in length, the Hsp90α mutation reduced piRNAs of 24-32 nucleotides, suggesting the presence of both FKBP6-dependent and -independent actions of HSP90α.This study revealed the specialized function of the HSP90α isofom in the piRNA biogenesis and repression of retrotransposons during the development of male germ cells in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Kita-ku, Okayama 700-8558, Japan Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.

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The Hsp90α mutation affected the localization but not the arginine methylation of MIWI2. (A) Immunofluorescence staining of HSP90α (green; left) or HSP90β (green; right) in WT E18.5 testes. Nuclei were counter-stained with DAPI (blue). (B) Localization of piRNA-related proteins (green) in E18.5 WT (top) and Hsp90α KO (bottom) testis. Bars: A: 50 μm B: 20 μm. (C) MIWI2 was immunoprecipitated from the E16.5 testes of WT or Hsp90α KO mice and subjected to western blotting. Immunoprecipitants were blotted for MIWI2 (top), methyl-arginine (middle) and WDR77 (bottom). See also Supplementary Figure S1.
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Figure 1: The Hsp90α mutation affected the localization but not the arginine methylation of MIWI2. (A) Immunofluorescence staining of HSP90α (green; left) or HSP90β (green; right) in WT E18.5 testes. Nuclei were counter-stained with DAPI (blue). (B) Localization of piRNA-related proteins (green) in E18.5 WT (top) and Hsp90α KO (bottom) testis. Bars: A: 50 μm B: 20 μm. (C) MIWI2 was immunoprecipitated from the E16.5 testes of WT or Hsp90α KO mice and subjected to western blotting. Immunoprecipitants were blotted for MIWI2 (top), methyl-arginine (middle) and WDR77 (bottom). See also Supplementary Figure S1.

Mentions: Immunofluorescence analysis revealed that HSP90α is specifically expressed in germ cells (prospermatogonia) in the testis on embryonic day 16.5 (E16.5), whereas HSP90β is expressed in both somatic and germ cells, suggesting a germ cell-specific function of HSP90α (Figure 1A). The prospermatogonia produce piRNAs at this developmental stage, and a deficiency in the biogenesis of fetal piRNAs in animals with mutations in piRNA-related genes such as Mili, Miwi2, Tdrd1, Tdrd9, Mov10l1, Maelstrom and Mitopld leads to a failure in spermatogenesis (10). We therefore investigated whether subcellular localization and/or expression levels of piRNA-related proteins are affected by the Hsp90α KO mutation in E18.5 testes. Proteins involved in piRNA biogenesis co-localize at granules around the periphery of the nucleus. MILI, one of the two PIWI proteins expressed in prospermatogonia, and TDRD1 localize at cytoplasmic granules at the periphery of nucleus, called pi-bodies, whereas the other PIWI protein MIWI2 as well as TDRD9 and MAELSTROM localizes at different cytoplasmic granules around the nucleus, called piP-bodies (28). MIWI2 and TDRD9 also localize in the nucleus, which is thought to be important for piRNA-targeted DNA methylation (25,29). In Hsp90α KO mice, MILI expression and localization were unaffected (Figure 1B and Supplementary Figure S1A). However, we detected an obvious difference in MIWI2 localization. Although MIWI2 localizes both at perinuclear granules and in the nucleus in the WT germ cells, nuclear staining was significantly decreased in Hsp90α KO germ cells (Figure 1B), although some staining still remained in nuclei. At the mRNA level, the Miwi2 expression was not affected in Hsp90α KO testes at E16.5 (Supplementary Figure S1B).


HSP90α plays an important role in piRNA biogenesis and retrotransposon repression in mouse.

Ichiyanagi T, Ichiyanagi K, Ogawa A, Kuramochi-Miyagawa S, Nakano T, Chuma S, Sasaki H, Udono H - Nucleic Acids Res. (2014)

The Hsp90α mutation affected the localization but not the arginine methylation of MIWI2. (A) Immunofluorescence staining of HSP90α (green; left) or HSP90β (green; right) in WT E18.5 testes. Nuclei were counter-stained with DAPI (blue). (B) Localization of piRNA-related proteins (green) in E18.5 WT (top) and Hsp90α KO (bottom) testis. Bars: A: 50 μm B: 20 μm. (C) MIWI2 was immunoprecipitated from the E16.5 testes of WT or Hsp90α KO mice and subjected to western blotting. Immunoprecipitants were blotted for MIWI2 (top), methyl-arginine (middle) and WDR77 (bottom). See also Supplementary Figure S1.
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Figure 1: The Hsp90α mutation affected the localization but not the arginine methylation of MIWI2. (A) Immunofluorescence staining of HSP90α (green; left) or HSP90β (green; right) in WT E18.5 testes. Nuclei were counter-stained with DAPI (blue). (B) Localization of piRNA-related proteins (green) in E18.5 WT (top) and Hsp90α KO (bottom) testis. Bars: A: 50 μm B: 20 μm. (C) MIWI2 was immunoprecipitated from the E16.5 testes of WT or Hsp90α KO mice and subjected to western blotting. Immunoprecipitants were blotted for MIWI2 (top), methyl-arginine (middle) and WDR77 (bottom). See also Supplementary Figure S1.
Mentions: Immunofluorescence analysis revealed that HSP90α is specifically expressed in germ cells (prospermatogonia) in the testis on embryonic day 16.5 (E16.5), whereas HSP90β is expressed in both somatic and germ cells, suggesting a germ cell-specific function of HSP90α (Figure 1A). The prospermatogonia produce piRNAs at this developmental stage, and a deficiency in the biogenesis of fetal piRNAs in animals with mutations in piRNA-related genes such as Mili, Miwi2, Tdrd1, Tdrd9, Mov10l1, Maelstrom and Mitopld leads to a failure in spermatogenesis (10). We therefore investigated whether subcellular localization and/or expression levels of piRNA-related proteins are affected by the Hsp90α KO mutation in E18.5 testes. Proteins involved in piRNA biogenesis co-localize at granules around the periphery of the nucleus. MILI, one of the two PIWI proteins expressed in prospermatogonia, and TDRD1 localize at cytoplasmic granules at the periphery of nucleus, called pi-bodies, whereas the other PIWI protein MIWI2 as well as TDRD9 and MAELSTROM localizes at different cytoplasmic granules around the nucleus, called piP-bodies (28). MIWI2 and TDRD9 also localize in the nucleus, which is thought to be important for piRNA-targeted DNA methylation (25,29). In Hsp90α KO mice, MILI expression and localization were unaffected (Figure 1B and Supplementary Figure S1A). However, we detected an obvious difference in MIWI2 localization. Although MIWI2 localizes both at perinuclear granules and in the nucleus in the WT germ cells, nuclear staining was significantly decreased in Hsp90α KO germ cells (Figure 1B), although some staining still remained in nuclei. At the mRNA level, the Miwi2 expression was not affected in Hsp90α KO testes at E16.5 (Supplementary Figure S1B).

Bottom Line: HSP90, found in all kingdoms of life, is a major chaperone protein regulating many client proteins.Whereas the mutation in Fkbp6 encoding a co-chaperone reduced piRNAs of 28-32 nucleotides in length, the Hsp90α mutation reduced piRNAs of 24-32 nucleotides, suggesting the presence of both FKBP6-dependent and -independent actions of HSP90α.This study revealed the specialized function of the HSP90α isofom in the piRNA biogenesis and repression of retrotransposons during the development of male germ cells in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Kita-ku, Okayama 700-8558, Japan Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.

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Related in: MedlinePlus