Microinjection of Antisense Oligonucleotides into Living Mouse Testis Enables LncRNA Function Study

Chen, Zhaohui; Li, Ling; Shi, Xiao-Lian; Li, Wu; Zhai, Huicong; Kang, Zhenlong; Zheng, Bangjin; Zhu, Jiaqi; Ye, Suni; Wang, Hao; Tong, Lingxiu; Huang, Chaoyang; Li, Yang; Zheng, Ke

doi:10.21203/rs.3.rs-707581/v1

Cited by 2 publications

(4 citation statements)

References 55 publications

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“…2e). To elucidate the in vivo function of Elfn2, we knocked down Elfn2 through testis microinjection of antisense oligonucleotides (ASOs), as we established recently 76 . We validated knockdown efficiency 11 days after ASO transduction to P35 testes by western blot analysis.…”

Section: Knockdown Of Endogenous Elfn2 Recapitulates Deficiencies Fro...mentioning

confidence: 99%

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Single-Cell RNA-seq Uncovers Dynamic Processes Orchestrated by RNA-Binding Protein DDX43 in Chromatin Remodeling during Spermiogenesis

Tan

Zhou

Wang

et al. 2022

Preprint

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Advances in single-cell RNA sequencing (scRNA-seq) have allowed for elucidating biological mechanisms at cell state level. Mammalian spermatogenic process showcases dynamic switches of gene expression pattern with delicate morphological and functional alterations of germ cells, but it is unclear how such dynamics is genetically controlled. Here we demonstrate that mouse testis-enriched RNA helicase DDX43, as well as its ATP hydrolysis site, is required for spermiogenesis. Genetic mutation of Ddx43 renders spermatids heterogeneously defective in multiple steps of chromatin remodeling, resulting in incomplete substitution of transition protein by protamine and less condensed sperm nucleus. Through scRNA-seq analyses of testicular cells derived from adult wild-type and Ddx43 mutant testes in mice, we reveal that the DDX43 deficiency-elicited perturbation in the dynamic RNA regulatory processes underlies the differentiation deficiency of spermatids. Further, focused analyses on early-stage spermatids combined with enhanced CLIP sequencing (eCLIP-seq) identify Elfn2 as DDX43-targeted hub gene, whose in vivo knockdownshows similar phenotypic defects as Ddx43 mutant. Our study illustrates an essential role for DDX43 in post-meiotic chromatin remodeling and highlights the single cell-based strategy for a refined dissection of stage-specific regulation of germline differentiation.

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Section: Knockdown Of Endogenous Elfn2 Recapitulates Deficiencies Fro...mentioning

confidence: 99%

“…We performed the transduction as previously described 76 . In brief, P24 mice were anesthetized by tri-bromoethanol.…”

Section: Aso-based Knockdown In Testismentioning

confidence: 99%

Single-Cell RNA-seq Uncovers Dynamic Processes Orchestrated by RNA-Binding Protein DDX43 in Chromatin Remodeling during Spermiogenesis

Tan

Zhou

Wang

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…2e). To elucidate the in vivo function of Elfn2, we knocked down Elfn2 through testis microinjection of antisense oligonucleotides (ASOs), as we established recently 77 . We validated knockdown efficiency 11 days after ASO transduction to P35 testes by western blot analysis.…”

Section: Knockdown Of Endogenous Elfn2 Recapitulates Deficiencies Fro...mentioning

confidence: 99%

“…We performed the transduction as previously described 77 . In brief, P24 mice were anesthetized by tri-bromoethanol.…”

Section: Aso-based Knockdown In Testismentioning

confidence: 99%

Single-Cell RNA-seq Uncovers Dynamic Processes Orchestrated by RNA-Binding Protein DDX43 in Chromatin Remodeling during Spermiogenesis

Zheng

Tan

Zhou

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Mammalian spermatogenesis shows prominent chromatin and transcriptomic switches with delicate morphological and functional alterations in germ cells, but it is unclear how such dynamics are controlled. Here we identify RNA helicase DDX43 as an essential regulator of the chromatin remodeling process during spermiogenesis. Testis-specific Ddx43 knockout mice show male infertility with defective histone-to-protamine replacement and post-meiotic chromatin condensation defects. In addition, the loss of its ATP hydrolysis activity by a missense mutation in the ATPase motif replicates the infertility phenotype in global Ddx43 knockout mice. Single-cell RNA sequencing (scRNA-seq) analyses of germ cells depleted of Ddx43 or expressing the Ddx43 ATPase-dead mutant reveals that DDX43 regulates dynamic RNA regulatory processes that underlie spermatid chromatin remodeling and differentiation. Transcriptomic profiling focusing on early-stage spermatids combined with enhanced crosslinking immunoprecipitation and sequencing (eCLIP-seq) further identify Elfn2 as DDX43-targeted hub gene. Elfn2 knockdown in testis leads to a phenotype similar to Ddx43 knockout or Ddx43 ATPase-dead knock-in. These findings illustrate an essential role for DDX43 in post-meiotic chromatin remodeling and highlight the single-cell-based strategy for a refined dissection of cell-state-specific regulation of germline development.

show abstract

Microinjection of Antisense Oligonucleotides into Living Mouse Testis Enables LncRNA Function Study

Cited by 2 publications

References 55 publications

Single-Cell RNA-seq Uncovers Dynamic Processes Orchestrated by RNA-Binding Protein DDX43 in Chromatin Remodeling during Spermiogenesis

Single-Cell RNA-seq Uncovers Dynamic Processes Orchestrated by RNA-Binding Protein DDX43 in Chromatin Remodeling during Spermiogenesis

Single-Cell RNA-seq Uncovers Dynamic Processes Orchestrated by RNA-Binding Protein DDX43 in Chromatin Remodeling during Spermiogenesis

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