Restricted and non-essential redundancy of RNAi and piRNA pathways in mouse oocytes

Abstract

Germline genome defense evolves to recognize and suppress retrotransposons. One of defensive mechanisms is the PIWI-associated RNA (piRNA) pathway, which employs small RNAs for sequence-specific post-transcriptional and transcriptional repression. The loss of the piRNA pathway in mice causes male sterility while females remain fertile. Unlike spermatogenic cells, mouse oocytes have also RNA interference (RNAi), another small RNA pathway capable of retrotransposon suppression. To examine whether RNAi compensates the loss of the piRNA pathway in mouse oocytes, we produced a new RNAi pathway mutant Dicer SOM and crossed it with a catalytically-dead mutant of Mili, an essential piRNA gene. Normal follicular and oocyte in double mutants showed that RNAi does not suppress a strong piRNA knock-out phenotype.However, we observed redundant and non-redundant targeting of specific retrotransposons.Intracisternal A Particle retrotransposon was mainly targeted by the piRNA pathway, MT and RLTR10 retrotransposons were targeted mainly by RNAi. Importantly, only double mutants showed increased background levels of transcripts potentially originating from intact LINE-1 elements. Our results thus show that while both small RNA pathways are simultaneously expendable defense pathways for ovarian oocyte development, yet another transcriptional silencing mechanism must mediate LINE-1 repression in female germ cells.3 Author summaryRetrotransposons are mobile genomic parasites causing mutations. Germ cells need protection against retrotransposons to prevent heritable transmission of their new insertions. The piRNA pathway is an ancient germline defense system analogous to acquired immunity: once a retrotransposons jumps into a specific "genomic checkpoint", it is recognized and suppressed.Remarkably, the murine piRNA pathway is essential for spermatogenesis but not oocyte development. In contrast, zebrafish lacking the piRNA pathway do not develop any germ cells. It was hypothesized that RNA interference pathway could rescue oocyte development in mice lacking the piRNA pathway. RNA interference also targets retrotransposons and is particularly enhanced in mouse oocytes. To test this hypothesis, we engineered mice lacking both pathways and observed that oocytes in these mice develop normally, which argues against the hypothesis.Furthermore, analysis of individual retrotransposon groups revealed that in specific cases the two pathways can mutually compensate each other. However, this ability is restricted to specific retrotransposon groups and seems to evolve stochastically. Finally, our results also indicate that there must be yet another layer of retrotransposon silencing in mouse oocytes, which prevents high retrotransposon activity in the absence of piRNA and RNA interference pathways.

Enhanced RNAi does not provide efficient innate antiviral immunity in micein vivo

“…6B) and differed from previously reported expression of CAG (Miyazaki et al, 1989;Okabe et al, 1997). The leakage was the highest in testes where it was observed in meiotic and postmeiotic cells (Taborska, 2024). Analysis of total Dicer mRNA expression in different organs of animals carrying one or two copies of the transgene suggested 1-3 times increased Dicer transcript level, except of the testis, where the leakage was much stronger (Fig.…”

“…The expression differs from the CAG promoter expression pattern observed in other transgenes (Buccheri et al, 2024;Nejepinska et al, 2012;Okabe et al, 1997). The western blot was adopted from (Taborska, 2024)…”

“…6A). This transgene was produced for Cre-inducible expression of C-terminally HA-tagged Dicer O (Dicer O-HA ) from the CAG promoter (Taborska, 2024). However, analysis of the transgenic mouse line revealed that the uninduced allele had leaky expression, which varied across organs (Fig.…”

“…Importantly, converting half of endogenous Dicer expression into the highly active Dicer variant in vivo has a negligible effect on canonical miRNAs but brings an order of magnitude more siRNAs and several mirtrons (a non-canonical miRNA class) across organs (Buccheri et al, 2024). Our secondary RNAi model was Dicer O expressed from a transgene inserted into the ROSA26 locus (Taborska, 2024). Although the second model was not working as originally designed, it delivered higher Dicer O expression and increased RNAi in multiple organs (Taborska, 2024).…”

“…Our secondary RNAi model was Dicer O expressed from a transgene inserted into the ROSA26 locus (Taborska, 2024). Although the second model was not working as originally designed, it delivered higher Dicer O expression and increased RNAi in multiple organs (Taborska, 2024).…”