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; Ni, Juan; Huang, Chaoyang; Li, Yang; Zheng, Ke

doi:10.1186/s13578-021-00717-y

Cited by 13 publications

(10 citation statements)

References 67 publications

(65 reference statements)

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“…LncRNAs have been reported to play important roles in reproduction and some lncRNAs have been explored by knockout mouse models and antisense oligonucleotide systems in vivo [ 29 – 31 ]. Antisense oligonucleotides of lncRNA Tsx were microinjected into seminiferous tubules and then promoted germ cell apoptosis through inhibiting lncRNA Tsx expression [ 29 ].…”

Section: Discussionmentioning

confidence: 99%

Interaction of lncRNA Gm2044 and EEF2 promotes estradiol synthesis in ovarian follicular granulosa cells

Hu,

Wang,

et al. 2023

J Ovarian Res

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The functions and molecular mechanisms of long noncoding RNA (lncRNA) in reproduction have been widely studied at present. However, lncRNA regulating hormone synthesis in ovarian follicular granulosa cells has not been sufficiently studied. Our previous research demonstrated that lncRNA Gm2044 could promote estradiol synthesis in follicular granulosa cells. In this study, we identified 21 binding proteins of lncRNA Gm2044 in ovarian follicles using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS). RNA immunoprecipitation (RNA IP) and reverse transcription PCR (RT-PCR) confirmed that lncRNA Gm2044 can interact with eukaryotic translation elongation factor 2 (EEF2) protein. Furthermore, we constructed lncRNA Gm2044 knockout mice using the CRISPR/Cas9 method. Although the follicular development and fertility of female lncRNA Gm2044 knockout mice were not affected, the serum estradiol concentration in female lncRNA Gm2044 knockout mice significantly decreased. Western blotting and ELISA revealed that lncRNA Gm2044 may promote the binding of EEF2 to Nr5a1 mRNA and then enhance the Nr5a1 mRNA translation, and the upregulated NR5A1 protein can strengthen estradiol synthesis. To determine the potential signaling pathway of lncRNA Gm2044 regulating estradiol synthesis, transcriptome sequencing was performed for ovaries of adult lncRNA Gm2044 knockout mice, which identified 565 significant up-regulated genes and 303 significant down-regulated genes, which were then analyzed with Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and validated by molecular experiments. Understanding how lncRNA Gm2044/EEF2 protein regulates estradiol synthesis will help treat estrogen-related reproductive diseases.

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Section: Discussionmentioning

confidence: 99%

Interaction of lncRNA Gm2044 and EEF2 promotes estradiol synthesis in ovarian follicular granulosa cells

Hu,

Wang,

et al. 2023

J Ovarian Res

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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: Resultsmentioning

confidence: 99%

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

Section: Methodsmentioning

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

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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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“…To the best of our knowledge, this is the first example of a multifunctional lncRNA in the testis. Since several lncRNAs were reported to be expressed in different cell types in the testis, with different subcellular localizations, or in different tissues including the testis [ 28 , 29 , 52 , 56 ], more testicular lncRNAs may be multifunctional. Our findings provide an insight into a further understanding of the multifunctionality of testis lncRNAs.…”

Section: Discussionmentioning

confidence: 99%

Evidence for a functional role of Start, a long noncoding RNA, in mouse spermatocytes

et al. 2022

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A mouse testis-specific long noncoding RNA (lncRNA), Start, is localized in the cytosol of Leydig cells and in the nucleus of pachytene spermatocytes. We previously showed that Start regulates steroidogenesis through controlling the expression of Star and Hsd3b1 genes in Leydig cells, but its function in germ cells was not known. Here we verified that a spermatocyte-specific protease gene, Prss43/Tessp-3, was downregulated in Start-knockout testes. To investigate the transcriptional regulatory activity of Start in spermatocytes, we first performed a series of reporter gene assays using a thymidine kinase promoter in spermatocyte-derived GC-2spd(ts) cells. A 5.4-kb genome sequence encompassing Start exhibited enhancer activity for this promoter, and the activity was decreased by knockdown of Start. Deletion of the Start promoter and replacement of the Start sequence abolished the enhancer activity and, consistently, the activity was detected in further experiments only when Start was actively transcribed. We then examined whether the Prss43/Tessp-3 gene could be a target of Start. A reporter gene assay demonstrated that the 5.4-kb sequence exhibited enhancer activity for a Prss43/Tessp-3 promoter in GC-2spd(ts) cells and that the activity was significantly decreased by knockdown of Start. These results suggest that Start functions in transcriptional activation of the Prss43/Tessp-3 gene in spermatocytes. Given that Start is presumed to regulate steroidogenic genes at the posttranscriptional level in Leydig cells, the function in spermatocytes is a novel role of Start. These findings provide an insight into multifunctionality of lncRNAs in the testis.

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Microinjection of antisense oligonucleotides into living mouse testis enables lncRNA function study

Cited by 13 publications

References 67 publications

Interaction of lncRNA Gm2044 and EEF2 promotes estradiol synthesis in ovarian follicular granulosa cells

Interaction of lncRNA Gm2044 and EEF2 promotes estradiol synthesis in ovarian follicular granulosa cells

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

Evidence for a functional role of Start, a long noncoding RNA, in mouse spermatocytes

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