Retrotransposons are "copy-and-paste" insertional mutagens that substantially contribute to mammalian genome content. Retrotransposons often carry long terminal repeats (LTRs) for retrovirus-like reverse transcription and integration into the genome. We report an extraordinary impact of a group of LTRs from the mammalian endogenous retrovirus-related ERVL retrotransposon class on gene expression in the germline and beyond. In mouse, we identified more than 800 LTRs from ORR1, MT, MT2, and MLT families, which resemble mobile gene-remodeling platforms that supply promoters and first exons. The LTR-mediated gene remodeling also extends to hamster, human, and bovine oocytes. The LTRs function in a stagespecific manner during the oocyte-to-embryo transition by activating transcription, altering protein-coding sequences, producing noncoding RNAs, and even supporting evolution of new protein-coding genes. These functions result, for example, in recycling processed pseudogenes into mRNAs or lncRNAs with regulatory roles. The functional potential of the studied LTRs is even higher, because we show that dormant LTR promoter activity can rescue loss of an essential upstream promoter. We also report a novel protein-coding gene evolution-D6Ertd527e-in which an MT LTR provided a promoter and the 5

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Mice lacking Cnot6l, a deadenylase component of the CCR4–NOT complex, are viable, but females have ∼40% smaller litters. Cnot6l is a maternal-effect gene acting in maternal mRNA degradation.

Abstract MicroRNAs (miRNAs) are ubiquitous small RNAs guiding post-transcriptional gene repression in countless biological processes. However, the miRNA pathway in mouse oocytes appears inactive and dispensable for development. We propose that marginalization of the miRNA pathway activity stems from the constraints and adaptations of RNA metabolism elicited by the diluting effects of oocyte growth. We report that miRNAs do not accumulate like mRNAs during the oocyte growth because miRNA turnover has not adapted to it. The most abundant miRNAs total tens of thousands of molecules in growing (∅ 40 μm) and fully grown (∅ 80 μm) oocytes, a number similar to that observed in much smaller fibroblasts. The lack of miRNA accumulation results in a 100-fold lower miRNA concentration in fully grown oocytes than in somatic cells. This brings a knock-down-like effect, where diluted miRNAs engage targets but are not abundant enough for significant repression. Low-miRNA concentrations were observed in rat, hamster, porcine and bovine oocytes, arguing that miRNA inactivity is not mouse-specific but a common mammalian oocyte feature. Injection of 250,000 miRNA molecules was sufficient to restore reporter repression in mouse and porcine oocytes, suggesting that miRNA inactivity comes from low-miRNA abundance and not from some suppressor of the pathway.

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.

RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.

PIWI-interacting RNAs (piRNAs) support the germline by suppressing retrotransposons. Studies of the pathway in mice have strongly shaped the view that mammalian piRNAs are essential for male but not for female fertility. Here, we report that the role of the piRNA pathway substantially differs in golden hamsters (Mesocricetus auratus), the piRNA pathway setup of which more closely resembles that of other mammals, including humans. The loss of the Mov10l1 RNA helicase—an essential piRNA biogenesis factor—leads to striking phenotypes in both sexes. In contrast to mice, female Mov10l1–/– hamsters are sterile because their oocytes do not sustain zygotic development. Furthermore, Mov10l1–/– male hamsters have impaired establishment of spermatogonia accompanied by transcriptome dysregulation and an expression surge of a young retrotransposon subfamily. Our results show that the mammalian piRNA pathway has essential roles in both sexes and its adaptive nature allows it to manage emerging genomic threats and acquire new critical roles in the germline.

Tens of thousands of rapidly evolving long non-coding RNA (lncRNA) genes have been identified, but functions were assigned to relatively few of them. The lncRNA contribution to the mouse oocyte physiology remains unknown. We report the evolutionary history and functional analysis of Sirena1, the most expressed lncRNA and the 10th most abundant poly(A) transcript in mouse oocytes. Sirena1 appeared in the common ancestor of mouse and rat and became engaged in two different post-transcriptional regulations. First, antisense oriented Elob pseudogene insertion into Sirena1 exon 1 is a source of small RNAs targeting Elob mRNA via RNA interference. Second, Sirena1 evolved functional cytoplasmic polyadenylation elements, an unexpected feature borrowed from translation control of specific maternal mRNAs. Sirena1 knock-out does not affect fertility, but causes minor dysregulation of the maternal transcriptome. This includes increased levels of Elob and mitochondrial mRNAs. Mitochondria in Sirena1−/− oocytes disperse from the perinuclear compartment, but do not change in number or ultrastructure. Taken together, Sirena1 contributes to RNA interference and mitochondrial aggregation in mouse oocytes. Sirena1 exemplifies how lncRNAs stochastically engage or even repurpose molecular mechanisms during evolution. Simultaneously, Sirena1 expression levels and unique functional features contrast with the lack of functional importance assessed under laboratory conditions.