Messenger RNA targeted by pachytene piRNAs is not an efficient substrate for piRNA production

ContributorsDowling, Mark
Number of pages23
Master program titleMaster in Biology, Option : Genetics, Development and Evolution
Imprimatur date2022-01-31

PIWI proteins and their associated small RNAs called PIWI-interacting RNAs (piRNAs) restrict transposon activity in animal gonads to ensure fertility. piRNA-guided PIWI endonuclease cleavage (slicer activity) is experimentally demonstrated to trigger phased piRNA biogenesis in the fly and mammalian embryonic germline. Mammalian adult germline has an abundant pool of the so-called pachytene piRNAs whose functions and biogenesis are mysterious. While PIWI slicer-dependent biogenesis is one of the proposed mechanisms, this is not experimentally tested. Here we target the endogenous Ythdc2 mRNA with an abundant pachytene piRNA by creating knock-in mice with either perfect or partially complementary binding site within its 3′ UTR. We created mice with either single or ten binding sites. Analysis of PIWI-associated complexes reveal the expected piRNA-guided targeting by MIWI-bound and MILI-bound pachytene piRNAs resulting in slicer cleavage fragments: a 17-mer or 19-mer by-product fragment and a secondary pre-piRNA molecule. Surprisingly, very few of such cleavage reactions result in mature piRNAs, as the cleavage products remain abundantly associated with the PIWI complexes. Unexpectedly and contrary to current knowledge of PIWI slicer activity, the partially complementary binding sites with a 2 nucleotides-bulge at the expected cleavage site did not prevent slicing. We propose that slicing of endogenous mRNAs may not result in phased piRNA biogenesis, probably due to the absence of Uridine at the 5′ end of secondary pre-piRNAs inhibiting the loading of the RNA molecules on a new Piwi protein.

Research group
Citation (ISO format)
DOWLING, Mark. Messenger RNA targeted by pachytene piRNAs is not an efficient substrate for piRNA production. 2022.
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Master thesis
  • PID : unige:158733

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Creation02/02/2022 12:59:00 PM
First validation02/02/2022 12:59:00 PM
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