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 repression. The loss of the piRNA pathway in mice causes male sterility while females remain fertile. Unlike spermatogenic cells, mouse oocytes posses also RNA interference (RNAi), another small RNA pathway capable of retrotransposon suppression. To examine whether RNAi compensates the loss of the piRNA pathway, 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 development in double mutants showed that RNAi does not suppress a strong ovarian piRNA knock-out phenotype. However, we observed redundant and non-redundant targeting of specific retrotransposon families illustrating stochasticity of recognition and targeting of invading retrotransposons. Intracisternal A Particle retrotransposon was mainly targeted by the piRNA pathway, MaLR and RLTR10 retrotransposons were targeted mainly by RNAi. Double mutants showed accumulations of LINE-1 retrotransposon transcripts. However, we did not find strong evidence for transcriptional activation and mobilization of retrotransposition competent LINE-1 elements suggesting that while both defense pathways are simultaneously expendable for ovarian oocyte development, yet another transcriptional silencing mechanism prevents mobilization of LINE-1 elements. Retrotransposons 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 retrotransposon jumps into a piRNA-producing locus, which provides a kind of a “genomic sensor” for actively transposing elements, 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 mutually compensate each other. However, this redundancy apparently evolved stochastically and is restricted to specific retrotransposon groups. Finally, our results 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.

Manganese (II) ions are essential for a variety of bacterial cellular processes. The transcription factor MntR is a metallosensor that regulates Mn²⁺ ion homeostasis in the bacterium Bacillus subtilis. Its DNA-binding affinity is increased by Mn²⁺ ion binding, allowing it to act as a transcriptional repressor of manganese import systems. Although experimentally well-researched, the molecular mechanism that regulates this process is still a puzzle. Computational simulations supported by circular dichroism (CD), differential scanning calorimetry (DSC) and native gel electrophoresis (native-PAGE) experiments were employed to study MntR structural and dynamical properties in the presence and absence of Mn²⁺ ions. The results of molecular dynamics (MD) simulations revealed that Mn²⁺ ion binding reduces the structural dynamics of the MntR protein and shifts the dynamic equilibrium towards the conformations adequate for DNA binding. Results of CD and DSC measurements support the computational results showing the change in helical content and stability of the MntR protein upon Mn²⁺ ion binding. Further, MD simulations show that Mn²⁺ binding induces polarization of the protein electrostatic potential, increasing the positive electrostatic potential of the DNA-binding helices in particular. In order to provide a deeper understanding of the changes in protein structure and dynamics due to Mn²⁺ binding, a mutant in which Mn²⁺ binding is mimicked by a cysteine bridge was constructed and also studied computationally and experimentally.

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. Author summary: Retrotransposons 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.