Tandem immuno-purification of affinity-tagged proteins from mouse testis extracts for MS analysis

Tani, Naoki; Tanno, Nobuhiro; Ishiguro, Kei‐ichiro

doi:10.1016/j.xpro.2022.101452

Cited by 8 publications

(10 citation statements)

References 14 publications

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“…We used two different buffer conditions: Tris-Triton buffer [20 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl 2 , 2 mM DTT, 0.1% TritonX-100] or Hepes-NP40 buffer [30 mM Hepes-KOH (pH 7.3), 150 mM KOAc, 5 mM MgOAc, 5 mM DTT, 0.1% NP40]. LC-MS/MS analysis was performed as following previous paper 76 . The immunoprecipitated proteins were run on 4-12 % SDS-PAGE gel by 1 cm from the well and stained with SimplyBlue (Thermo Fisher) for in-gel digestion.…”

Section: Methodsmentioning

confidence: 99%

Mettl1-dependent m⁷G tRNA modification is essential for maintaining spermatogenesis and fertility inDrosophila melanogaster

Kaneko,

Miyoshi,

Tomuro

et al. 2023

Preprint

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N7-methylguanosine (m7G) in the variable loop region of tRNA is catalyzed by METTL1/WDR4 heterodimer and stabilizes target tRNA. Here, we reveal essential functions of Mettl1 in Drosophila fertility. Knockout of Mettl1 (Mettl1-KO) lost the elongated spermatids and mature sperm, which was fully rescued by a Mettl1-transgene expression, but not a catalytic-dead Mettl1 transgene. This demonstrates that Mettl1-dependent m7G is required for spermatogenesis. Mettl1-KO resulted in a loss of m7G modification on a subset of tRNAs and a decreased level of tRNA expression. Strikingly, overexpression of the translational elongation factor, EF1α1, which can compete with the rapid tRNA decay (RTD) pathway in S. cerevisiae, significantly counteracted the sterility of Mettl1-KO males, supporting a critical role of m7G modification of tRNAs in spermatogenesis. Ribosome profiling showed that Mettl1-KO led to the ribosome stalling at codons decoded by tRNAs that were reduced in expression. Mettl1-KO also significantly reduced the translation efficiency of genes involved in elongated spermatid formation and sperm stability. These findings reveal a developmental role for m7G tRNA modifications and indicate that m7G modification-dependent tRNA stability differs among tissues.

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Section: Methodsmentioning

confidence: 99%

Mettl1-dependent m⁷G tRNA modification is essential for maintaining spermatogenesis and fertility inDrosophila melanogaster

Kaneko,

Miyoshi,

Tomuro

et al. 2023

Preprint

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“…Testis chromatin-bound and -unbound extracts were prepared as described previously (Tani et al 2022). Briefly, testes were removed from male C57BL/6 mice (P18-23, 4-weeks and 8-weeks old), detunicated, and then resuspended in low salt extraction buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 0.4 mM EDTA, 0.1% TritonX100, 10% glycerol, 1 mM β-mercaptoethanol) supplemented with Complete Protease Inhibitor (Roche).…”

Section: Methods Detailsmentioning

confidence: 99%

“…Testis chromatin-bound and -unbound extracts were prepared as described previously (Tani et al 2022). Briefly, testes were removed from male C57BL/6 mice (P18-23, 4-…”

Section: Preparation Of Testis Extracts and Immunoprecipitationmentioning

confidence: 99%

“…MEIOSIN, together with STRA8 (Kojima et al2019), activates numerous meiotic genes. Among the target genes of MEIOSIN whose functions are unknown, we screened new germ cell-specific factors involved in meiosis (Takemoto et al 2020) (Horisawa-Takada et al 2021) (Tanno et al 2022). In the present study, we identified the Heat shock transcription factor family 5 (Hsf5) gene that encodes a HSF as one of the MEIOSIN/STRA8 target genes.…”

Section: Introductionmentioning

confidence: 99%

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Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions

Yoshimura,

Shimada,

Kikuchi

et al. 2023

Preprint

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Meiotic prophase progression is differently regulated in males and females. In males, pachytene transition during meiotic prophase is accompanied by robust alteration in gene expression. However, how gene expression is regulated differently to ensure meiotic prophase completion in males remains elusive. Herein, we identified HSF5 as a male germ cell-specific heat shock transcription factor (HSF) for meiotic prophase progression. Genetic analyses and single-cell RNA-sequencing demonstrated that HSF5 is essential for progression beyond the pachytene stage under non-stress conditions rather than heat stress. Chromatin binding analysis in vivo and DNA-binding assays in vitro suggested that HSF5 binds to promoters in a subset of genes associated with chromatin organization. HSF5 recognizes a DNA motif different from typical heat shock elements recognized by other canonical HSFs. This study suggests that HSF5 is an atypical HSF that enforces the gene expression program for pachytene transition during meiotic prophase in males.

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“… Note: Some tissue samples may be challenging to homogenize with glass beads. Alternative methods can be used ( Jenkins et al., 2020 ; Tani et al., 2022 ; van Smeden et al., 2011 ). Mouse serum.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Protocol for rapid and accurate quantification of phospholipids in yeast and mammalian systems using LC-MS

Yang

Xue

2022

STAR Protocols

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Tandem immuno-purification of affinity-tagged proteins from mouse testis extracts for MS analysis

Cited by 8 publications

References 14 publications

Mettl1-dependent m⁷G tRNA modification is essential for maintaining spermatogenesis and fertility inDrosophila melanogaster

Mettl1-dependent m⁷G tRNA modification is essential for maintaining spermatogenesis and fertility inDrosophila melanogaster

Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions

Protocol for rapid and accurate quantification of phospholipids in yeast and mammalian systems using LC-MS

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Tandem immuno-purification of affinity-tagged proteins from mouse testis extracts for MS analysis

Cited by 8 publications

References 14 publications

Mettl1-dependent m7G tRNA modification is essential for maintaining spermatogenesis and fertility inDrosophila melanogaster

Mettl1-dependent m7G tRNA modification is essential for maintaining spermatogenesis and fertility inDrosophila melanogaster

Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions

Protocol for rapid and accurate quantification of phospholipids in yeast and mammalian systems using LC-MS

Contact Info

Product

Resources

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Mettl1-dependent m⁷G tRNA modification is essential for maintaining spermatogenesis and fertility inDrosophila melanogaster

Mettl1-dependent m⁷G tRNA modification is essential for maintaining spermatogenesis and fertility inDrosophila melanogaster