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| Status |
Public on Oct 23, 2023 |
| Title |
In vivo PIWI slicing in mouse testes deviates from rules established in vitro |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Purified endogenous mouse MIWI fails to cleave mismatched targets in vitro. Surprisingly, here we find using knock-in mouse models that piRNA target sites with cleavage-site mismatches are precisely sliced in vivo. This is identical to the slicing outcome in knock-in mice where targets are identified by perfect complementarity base-pairing with the piRNA. Additionally, we find that such pachytene piRNA-guided MIWI/MILI slicing in vivo failed to initiate phased piRNA production from the specific target mRNA we studied. Instead, the two slicer cleavage fragments were retained in PIWI proteins as a pre-piRNA and 17-19 nt by-product fragments. Our results indicate that PIWI slicing rules established in vitro are not respected in vivo, and that all targets of PIWI slicing are not substrates for piRNA biogenesis.
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| Overall design |
We designed four knock-in mouse lines: two lines with one or ten sites perfectly complementary to the pachytene piRNA piR-A (1xPerf, 10xPerf) and two lines with one or ten sites having mismatches at positions 10-11 (1xBulge,10xBulge). The piRNA targeted sites were inserted into 3′UTR of Ythdc2. We immunoprecipitated and sequenced the MILI and MIWI associated small RNAs to search for piRNAs generated by piR-A cleavage and used RNA-seq to analyze the gene expression changes. Two biological replicas were analyzed.
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| Contributor(s) |
Dowling M, Raad N, Homolka D, Gos P, Raman Pandey R, Pillai RS |
| Citation(s) |
36617658 |
| |
| Submission date |
Dec 01, 2022 |
| Last update date |
Oct 24, 2023 |
| Contact name |
Ramesh Pillai |
| E-mail(s) |
ramesh.pillai@unige.ch
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| Organization name |
University of Geneva
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| Department |
Department of Molecular Biology
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| Street address |
30, Quai Ernest-Ansermet
|
| City |
Gneveva |
| ZIP/Postal code |
CH-1211 |
| Country |
Switzerland |
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| Platforms (2) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (26)
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| Relations |
| BioProject |
PRJNA907392 |
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