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Dazl regulates germ cell survival through a network of polA-proximal mRNA interactions [GC1-spg cells (parental line) Ribosome Profiling and RNA-seq]

ABSTRACT: The RNA binding protein Dazl is essential for gametogenesis, but its direct in vivo functions, RNA targets, and the molecular basis for germ cell loss in DAZL null mice are unknown. Here, we mapped transcriptome-wide Dazl-RNA interactions in vivo, revealing Dazl binding to thousands of mRNAs via polyA-proximal 3?UTR interactions. In parallel, fluorescence activated cell sorting and RNA-Seq identified mRNAs sensitive to Dazl deletion in male germ cells. Despite binding a broad set of mRNAs, integrative analyses indicate that Dazl post-transcriptionally controls only a subset of its mRNA targets, namely those corresponding to a network of genes critical for germ cell proliferation and survival. Additionally, we provide evidence that polyA sequences have key roles in specifying Dazl-RNA interactions across the transcriptome. Altogether, our results reveal a mechanism for Dazl-RNA binding, and illustrate that Dazl functions as a master regulator of a post-transcriptional mRNA program essential for germ cell survival.

ORGANISM(S): Mus musculus

PROVIDER: GSE120097 | GEO | 2018/09/18

REPOSITORIES: GEO

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Dazl regulates germ cell survival through a network of polA-proximal mRNA interactions [GC1-spg cells (parental line) iCLIP]

Project description:The RNA binding protein Dazl is essential for gametogenesis, but its direct in vivo functions, RNA targets, and the molecular basis for germ cell loss in DAZL null mice are unknown. Here, we mapped transcriptome-wide Dazl-RNA interactions in vivo, revealing Dazl binding to thousands of mRNAs via polyA-proximal 3’UTR interactions. In parallel, fluorescence activated cell sorting and RNA-Seq identified mRNAs sensitive to Dazl deletion in male germ cells. Despite binding a broad set of mRNAs, integrative analyses indicate that Dazl post-transcriptionally controls only a subset of its mRNA targets, namely those corresponding to a network of genes critical for germ cell proliferation and survival. Additionally, we provide evidence that polyA sequences have key roles in specifying Dazl-RNA interactions across the transcriptome. Altogether, our results reveal a mechanism for Dazl-RNA binding, and illustrate that Dazl functions as a master regulator of a post-transcriptional mRNA program essential for germ cell survival.

2018-09-18 | GSE120098 | GEO

Dazl maintains proliferating germ cells through a network of polyA-proximal mRNA interactions [P6 iCLIP]

Project description:Dazl (deleted in azoospermia like) is a member of the DAZ family of germ cell-restricted RNA binding proteins required for gametogenesis from worm to human. The direct RNA targets and functions of these essential proteins are poorly understood. Here, we generated high-resolution, transcriptome-wide maps of Dazl-RNA interactions in mouse testes. These maps provide important insights into the mechanism of Dazl recruitment to mRNA and reveal Dazl binding to thousands of mRNAs predominantly through sequence-specific interactions near the polyA tail. Using transgenic mice and fluorescence activated cell sorting (FACS), we isolated DAZL knockout germ cells and used RNA-Seq to identify mRNAs sensitive to DAZL-ablation. Intersecting the RNA-Seq and Dazl-RNA interaction datasets revealed that Dazl enhances expression of a subset of directly-bound transcripts, namely mRNAs for a network of essential cell cycle regulatory genes. Collectively, our integrative analysis delineates a Dazl-dependent post-transcriptional gene regulatory program essential for mammalian germ cell maintenance.

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Dazl maintains proliferating germ cells through a network of polyA-proximal mRNA interactions [Spermatogonia PolyA-Seq]

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Dazl maintains proliferating germ cells through a network of polyA-proximal mRNA interactions [Adult RNA-Seq]

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Dazl maintains proliferating germ cells through a network of polyA-proximal mRNA interactions [Adult PolyA-Seq]

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Dazl maintains proliferating germ cells through a network of polyA-proximal mRNA interactions [Adult HITS-CLIP]

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Dazl maintains proliferating germ cells through a network of polyA-proximal mRNA interactions [GFP cells RNA-Seq]

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An RNA-interacting protein network regulates pluripotency in mammalian primordial germ cells (Dazl UV RNA-IP)

Project description:Primordial germ cells (PGCs) are the embryonic precursors to egg and sperm. When removed from the embryonic gonad, PGCs can give rise to embryonic germ cell lines (EGs), pluripotent stem cells that display most of the characteristics of embryonic stem cells (ESCs) including the ability to form teratomas and to contribute to chimera formation. In mice, EG cells can be derived between E8.5 up to E12.5 of embryonic development, at which point the PGCs undergo sexual differentiation and in the male transition into unipotent gonocytes. Dazl, a germ cell-specific RNA-binding protein, is specifically expressed in developing PGCs and is required for proper germ cell development. Dazl knockout mice are infertile, but the molecular mechanisms underlying this phenotype are still unknown. Here we demonstrate that Dazl localizes in granular structures in mammalian PGCs but not in ESCs. We demonstrate Dazl plays a central role in a large mRNA/protein interactive network that includes members of Fragile-X family RNA-binding proteins. We demonstrate that Dazl and Fxr1 play a central role in these granules and directly regulate the translation of specific core pluripotency factors, including Sox2 and Suz12. RNA species interacting specifically with Dazl in primordial germ cells were identified by RNA-IP microarray analysis. This dataset contains data from UV-crosslinked RNA-IPs from in vitro PGC lysates. UV-crosslinked RNA-IP (anti-GFP and anti-PABP1) experiments from in vitro derived primordial germ cells expressing Dazl-GFP-V5. Input Total RNA and polyA-binding protein 1 (PABP1) IP were used as controls. Control and IP-enriched RNA samples were analyzed by Agilent Mouse Whole Genome 4X44K one-color microarrays. Two replicates for each condition were done.

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Project description:The autosomal gene Dazl is a member of highly conservative DAZ family, all of which contains a RNA binding domain and expresses in germ cells. In mice, Dazl plays an essential role in germ cell development, germ cell lost in Dazl knockout mouse on pure C57BL/6 strain background. Here we use HITS-CLIP to globally characterize the genome-wide target RNA of Dazl in mouse testes. Sequencing data provides large quantities of clues to help us to get close to the truth of Dazl function. We find over 1700 RNAs are bind by Dazl mainly at 3’UTR and the genes associated with transcription, RNA splicing and reproductive cellular process are enriched significantly.

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RNA-seq of 10dpp wild type and Dazl SKO mouse testes

Project description:The autosomal gene Dazl is a member of highly conservative DAZ family, all of which contains a RNA binding domain and expresses in germ cells. In mice, Dazl plays an essential role in germ cell development, germ cell lost in Dazl knockout mouse on pure C57BL/6 strain background. Here we generated conditional DAZL knockout mice which allowed us to examine the roles of DAZL in postnatal spermatogenesis without affecting its early function in PGC. RNA was extracted from 10dpp wild type and Stra8-cre KO mouse testes to perform RNA-seq, StringTie software was used to assess the expression level by formula log2(FPKM+1).

2019-10-09 | GSE114042 | GEO

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