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| Status |
Public on Sep 01, 2013 |
| Title |
Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline |
| Organism |
Mus musculus |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
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| Summary |
Piwi proteins and their associated piRNAs are essential in the germline where they repress transposition, regulate translation, and guide epigenetic programming. Little is known, however, about the molecular mechanisms through which Piwi proteins and piRNAs mediate these processes. Here, we show that an evolutionarily conserved Tudor and KH-domain containing protein, Tdrkh (a.k.a. Tdrd2), partners with Miwi and Miwi2 in mice via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is localized to pi-bodies and piP-bodies and is required for nuclear localization of Miwi2. Genetic deletion of Tdrkh arrests meiosis at the zygotene stage, demethylates Line1 DNA, and up-regulates Line1 transposition, but does not promote apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs. Specifically, in Tdrkh mutants, piRNAs accumulate as a distinct population of 5’U-containing 31-37nt RNA that largely complements the missing mature piRNAs. These results demonstrate that the primary piRNA biogenesis pathway involves 3à5’ processing of the 31-37nt intermediates and that Tdrkh is required for this final step of piRNA biogenesis. However, Tdrkh is not required for the secondary piRNA biogenesis pathway (i.e., the ping pong cycle). These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent.
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| Overall design |
Tdrkh-floxed mice were generated by the University of Connecticut Gene Targeting and Transgenic Facility. The targeting vector utilized C57Bl6 Tdrkh genome sequences from BAC clone RP23-263K17 (Chori BACPAC) and floxed exons 2-4 (the start codon is in exon 2) and was electroporated into D2 ES cells, a male hybrid C57Bl6/129SEV line. Clones that survived positive selection with G418 and negative selection with gancyclovir were expanded and screened by PCR with primers recognizing endogenous and exogenous (vector-derived) sequences. Positive clones were fused to CD-1 embryos, and germline transmission from resulting pups was confirmed by test crosses to CD-1 mice (which also confirmed that all gametes were derived from the targeted clones). Positive animals were bred to FLP mice to delete the neomycin-targeting cassette, resulting in Tdrkh cKO mice on a C57Bl6/129 background. cKO mice were bred with EIIa-Cre transgenic mice to excise the floxed exons 2-4 and generate Tdrkh +/- animals. Heterozygous animas were intercrossed to remove the EIIA transgene. Two independent knockout lines were generated from independent cKO founder lines and showed identical phenotypes in all experiments performed.
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| Contributor(s) |
Lin H, Saxe J, Chen M |
| Citation(s) |
23714778 |
| Submission date |
May 21, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Mengjie Chen |
| E-mail(s) |
mengjie.chen@yale.edu
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| Organization name |
Yale University
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| Street address |
13 Pleasant St
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| City |
New Haven |
| ZIP/Postal code |
06511 |
| Country |
USA |
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| Platforms (1) |
| GPL9250 |
Illumina Genome Analyzer II (Mus musculus) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA204938 |
| SRA |
SRP022959 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE47151_RAW.tar |
139.8 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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