Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in DicerΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. DicerΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo.

• Klíčová slova
• Dicer, Mirtron, PKR, dsRNA, siRNA,
• MeSH
• DEAD-box RNA-helikasy genetika   metabolismus   MeSH
• dvouvláknová RNA * metabolismus   genetika   MeSH
• malá interferující RNA * genetika   metabolismus   MeSH
• mikro RNA genetika   metabolismus   MeSH
• myši MeSH
• protein - isoformy genetika   metabolismus   MeSH
• ribonukleasa III * genetika   metabolismus   MeSH
• RNA interference * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• Dicer1 protein, mouse MeSH Prohlížeč
• dvouvláknová RNA * MeSH
• malá interferující RNA * MeSH
• mikro RNA MeSH
• protein - isoformy MeSH
• ribonukleasa III * MeSH

MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer•-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.

• Klíčová slova
• DExD, Dicer, PKR, RNAi, TARBP2, cryo-EM, dsRBD, dsRNA, helicase, miRNA, mirtron,
• MeSH
• mikro RNA * genetika   metabolismus   MeSH
• myši MeSH
• ribonukleasa III * metabolismus   MeSH
• RNA interference MeSH
• savci metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA * MeSH
• ribonukleasa III * MeSH
• transportní proteiny MeSH

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.

• MeSH
• křečci praví MeSH
• křeček rodu Mesocricetus metabolismus   MeSH
• malá interferující RNA genetika   MeSH
• oocyty metabolismus   patologie   MeSH
• retroelementy fyziologie   MeSH
• RNA-helikasy genetika   MeSH
• spermatogeneze genetika   fyziologie   MeSH
• spermatogonie metabolismus   patologie   MeSH
• testis metabolismus   MeSH
• umlčování genů fyziologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá interferující RNA MeSH
• retroelementy MeSH
• RNA-helikasy MeSH

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.

• MeSH
• buňky 3T3 MeSH
• druhová specificita MeSH
• křečci praví MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• messenger RNA genetika   metabolismus   MeSH
• mikro RNA genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• oogeneze * MeSH
• prasata MeSH
• skot MeSH
• teoretické modely MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• krysa rodu Rattus MeSH
• myši MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• mikro RNA MeSH

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.

• MeSH
• genový knockout MeSH
• krysa rodu Rattus MeSH
• messenger RNA genetika   MeSH
• mitochondrie genetika   ultrastruktura   MeSH
• myši MeSH
• oocyty růst a vývoj   metabolismus   ultrastruktura   MeSH
• polyadenylace genetika   MeSH
• RNA dlouhá nekódující genetika   MeSH
• RNA mitochondriální genetika   MeSH
• transkriptom genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• mitochondrial messenger RNA MeSH Prohlížeč
• RNA dlouhá nekódující MeSH
• RNA mitochondriální MeSH

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 DicerSOM 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.

• MeSH
• Argonaut proteiny genetika   MeSH
• DEAD-box RNA-helikasy genetika   MeSH
• malá interferující RNA genetika   MeSH
• mutace MeSH
• myši MeSH
• oocyty chemie   růst a vývoj   MeSH
• retroelementy * MeSH
• ribonukleasa III genetika   MeSH
• RNA interference * MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Argonaut proteiny MeSH
• DEAD-box RNA-helikasy MeSH
• Dicer1 protein, mouse MeSH Prohlížeč
• malá interferující RNA MeSH
• Piwil2 protein, mouse MeSH Prohlížeč
• retroelementy * MeSH
• ribonukleasa III MeSH

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.

• MeSH
• buňky NIH 3T3 MeSH
• DEAD-box RNA-helikasy metabolismus   MeSH
• dvouvláknová RNA genetika   metabolismus   MeSH
• genový knockout MeSH
• kinasa eIF-2 genetika   MeSH
• malá interferující RNA metabolismus   MeSH
• mikro RNA metabolismus   MeSH
• myši MeSH
• plazmidy genetika   MeSH
• proteiny vázající RNA metabolismus   MeSH
• ribonukleasa III metabolismus   MeSH
• RNA interference fyziologie   MeSH
• sekvence nukleotidů genetika   MeSH
• transfekce MeSH
• transportní proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• Dicer1 protein, mouse MeSH Prohlížeč
• dvouvláknová RNA MeSH
• kinasa eIF-2 MeSH
• malá interferující RNA MeSH
• mikro RNA MeSH
• protein kinase R, mouse MeSH Prohlížeč
• proteiny vázající RNA MeSH
• Rbbp6 protein, mouse MeSH Prohlížeč
• ribonukleasa III MeSH
• trans-activation responsive RNA-binding protein MeSH Prohlížeč
• transportní proteiny MeSH

The oocyte-to-embryo transition (OET) arguably initiates with formation of a primordial follicle and culminates with reprogramming of gene expression during the course of zygotic genome activation. This transition results in converting a highly differentiated cell, i.e. oocyte, to undifferentiated cells, i.e. initial blastomeres of a preimplantation embryo. A plethora of changes occur during the OET and include, but are not limited to, changes in transcription, chromatin structure, and protein synthesis; accumulation of macromolecules and organelles that will comprise the oocyte's maternal contribution to the early embryo; sequential acquisition of meiotic and developmental competence to name but a few. This review will focus on transcriptional and post-transcriptional changes that occur during OET in mouse because such changes are likely the major driving force for OET. We often take a historical and personal perspective, and highlight how advances in experimental methods often catalyzed conceptual advances in understanding the molecular bases for OET. We also point out questions that remain open and therefore represent topics of interest for future investigation.

• MeSH
• buněčná diferenciace fyziologie   MeSH
• embryonální vývoj fyziologie   MeSH
• genom MeSH
• myši MeSH
• oocyty fyziologie   MeSH
• ovariální folikul fyziologie   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH

Small RNA molecules regulating gene expression received a status of omnipresent master regulators of eukaryotic lives with almost supernatural powers. Mammals hold at least three mechanisms employing small RNA molecules for regulating gene expression. One of these mechanisms, the microRNA (miRNA) pathway, involves currently over a thousand of genome-encoded different miRNAs that are claimed to extend their control over more than a half of a genome. Here, I discuss how and why mouse oocytes and early embryos ignore the regulatory power of miRNAs, adding another surprising feature to the field of small RNAs.

• MeSH
• embryo savčí metabolismus   MeSH
• mikro RNA genetika   MeSH
• myši MeSH
• oocyty metabolismus   MeSH
• RNA interference * MeSH
• RNA messenger skladovaná MeSH
• vývojová regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA MeSH
• RNA messenger skladovaná MeSH

RNA silencing is a complex of mechanisms that regulate gene expression through small RNA molecules. The microRNA (miRNA) pathway is the most common of these in mammals. Genome-encoded miRNAs suppress translation in a sequence-specific manner and facilitate shifts in gene expression during developmental transitions. Here, we discuss the role of miRNAs in oocyte-to-zygote transition and in the control of pluripotency. Existing data suggest a common principle involving miRNAs in defining pluripotent and differentiated cells. RNA silencing pathways also rapidly evolve, resulting in many unique features of RNA silencing in different taxonomic groups. This is exemplified in the mouse model of oocyte-to-zygote transition, in which the endogenous RNA interference pathway has acquired a novel role in regulating protein-coding genes, while the miRNA pathway has become transiently suppressed.

• MeSH
• fylogeneze MeSH
• lidé MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• mikro RNA klasifikace   genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• oocyty cytologie   fyziologie   MeSH
• pluripotentní kmenové buňky cytologie   fyziologie   MeSH
• RNA interference * MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• zvířata MeSH
• zygota cytologie   fyziologie   MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• malá interferující RNA MeSH
• mikro RNA MeSH