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1.
Mol Cell ; 82(21): 4064-4079.e13, 2022 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-36332606

RESUMO

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.


Assuntos
MicroRNAs , Ribonuclease III , Camundongos , Animais , Ribonuclease III/metabolismo , Interferência de RNA , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Transporte/metabolismo , Mamíferos/metabolismo
2.
Mol Cell ; 67(6): 1059-1067.e4, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28867294

RESUMO

YTHDF2 binds and destabilizes N6-methyladenosine (m6A)-modified mRNA. The extent to which this branch of m6A RNA-regulatory pathway functions in vivo and contributes to mammalian development remains unknown. Here we find that YTHDF2 deficiency is partially permissive in mice and results in female-specific infertility. Using conditional mutagenesis, we demonstrate that YTHDF2 is autonomously required within the germline to produce MII oocytes that are competent to sustain early zygotic development. Oocyte maturation is associated with a wave of maternal RNA degradation, and the resulting relative changes to the MII transcriptome are integral to oocyte quality. The loss of YTHDF2 results in the failure to regulate transcript dosage of a cohort of genes during oocyte maturation, with enrichment observed for the YTHDF2-binding consensus and evidence of m6A in these upregulated genes. In summary, the m6A-reader YTHDF2 is an intrinsic determinant of mammalian oocyte competence and early zygotic development.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Meiose , Oócitos/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Transcriptoma , Zigoto/metabolismo , Animais , Sítios de Ligação , Feminino , Fertilidade , Genótipo , Infertilidade Feminina/genética , Infertilidade Feminina/metabolismo , Infertilidade Feminina/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/patologia , Fenótipo , Ligação Proteica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Zigoto/patologia
3.
RNA ; 28(6): 842-853, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35304421

RESUMO

Long noncoding RNAs (lncRNAs) are rapidly evolving and thus typically poorly conserved in their sequences. How these sequence differences affect the characteristics and potential functions of lncRNAs with shared synteny remains unclear. Here we show that the syntenically conserved lncRNA Firre displays distinct expression and localization patterns in human and mouse. Single molecule RNA FISH reveals that in a range of cell lines, mouse Firre (mFirre) is predominantly nuclear, while human FIRRE (hFIRRE) is distributed between the cytoplasm and nucleus. This localization pattern is maintained in human/mouse hybrid cells expressing both human and mouse Firre, implying that the localization of the lncRNA is species autonomous. We find that the majority of hFIRRE transcripts in the cytoplasm are comprised of isoforms that are enriched in RRD repeats. We furthermore determine that in various tissues, mFirre is more highly expressed than its human counterpart. Our data illustrate that the rapid evolution of syntenic lncRNAs can lead to variations in lncRNA localization and abundance, which in turn may result in disparate lncRNA functions even in closely related species.


Assuntos
RNA Longo não Codificante , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
4.
Development ; 147(12)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32439762

RESUMO

Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of MLL3 and MLL4 mutations in many types of human cancer. Despite this emerging importance, the requirements of these paralogs in mammalian development have only been incompletely reported. Here, we examined the null phenotypes to establish that MLL3 is first required for lung maturation, whereas MLL4 is first required for migration of the anterior visceral endoderm that initiates gastrulation in the mouse. This collective cell migration is preceded by a columnar-to-squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, Mll4 mutants display incompletely penetrant, sex-distorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these paralogs in mouse development argues against their action as general enhancer factors.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Anormalidades Múltiplas/veterinária , Alelos , Animais , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Face/anormalidades , Face/patologia , Feminino , Genótipo , Doenças Hematológicas/genética , Doenças Hematológicas/patologia , Doenças Hematológicas/veterinária , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mutagênese , Gravidez , Insuficiência Respiratória/etiologia , Fatores de Tempo , Doenças Vestibulares/genética , Doenças Vestibulares/patologia , Doenças Vestibulares/veterinária
5.
Nature ; 548(7667): 347-351, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28792939

RESUMO

A fundamental principle in biology is that the program for early development is established during oogenesis in the form of the maternal transcriptome. How the maternal transcriptome acquires the appropriate content and dosage of transcripts is not fully understood. Here we show that 3' terminal uridylation of mRNA mediated by TUT4 and TUT7 sculpts the mouse maternal transcriptome by eliminating transcripts during oocyte growth. Uridylation mediated by TUT4 and TUT7 is essential for both oocyte maturation and fertility. In comparison to somatic cells, the oocyte transcriptome has a shorter poly(A) tail and a higher relative proportion of terminal oligo-uridylation. Deletion of TUT4 and TUT7 leads to the accumulation of a cohort of transcripts with a high frequency of very short poly(A) tails, and a loss of 3' oligo-uridylation. By contrast, deficiency of TUT4 and TUT7 does not alter gene expression in a variety of somatic cells. In summary, we show that poly(A) tail length and 3' terminal uridylation have essential and specific functions in shaping a functional maternal transcriptome.


Assuntos
Herança Materna/genética , Oócitos/metabolismo , Poli A/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma , Uridina Monofosfato/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Infertilidade Feminina/genética , Masculino , Camundongos , Camundongos Knockout , Mães , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Oócitos/crescimento & desenvolvimento , Especificidade de Órgãos , Poli A/química , Estabilidade de RNA
6.
PLoS Genet ; 12(6): e1006095, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27254021

RESUMO

Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.


Assuntos
Citoplasma/metabolismo , RNA Helicases DEAD-box/metabolismo , Ribonuclease III/metabolismo , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/genética , Dano ao DNA/genética , Camundongos , MicroRNAs/genética , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , Ribonuclease III/genética
7.
PLoS Genet ; 10(10): e1004597, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25329700

RESUMO

Male fertility requires the continuous production of high quality motile spermatozoa in abundance. Alterations in all three metrics cause oligoasthenoteratozoospermia, the leading cause of human sub/infertility. Post-mitotic spermatogenesis inclusive of several meiotic stages and spermiogenesis (terminal spermatozoa differentiation) are transcriptionally inert, indicating the potential importance for the post-transcriptional microRNA (miRNA) gene-silencing pathway therein. We found the expression of miRNA generating enzyme Dicer within spermatogenesis peaks in meiosis with critical functions in spermatogenesis. In an expression screen we identified two miRNA loci of the miR-34 family (miR-34b/c and miR-449) that are specifically and highly expressed in post-mitotic male germ cells. A reduction in several miRNAs inclusive of miR-34b/c in spermatozoa has been causally associated with reduced fertility in humans. We found that deletion of both miR34b/c and miR-449 loci resulted in oligoasthenoteratozoospermia in mice. MiR-34bc/449-deficiency impairs both meiosis and the final stages of spermatozoa maturation. Analysis of miR-34bc-/-;449-/- pachytene spermatocytes revealed a small cohort of genes deregulated that were highly enriched for miR-34 family target genes. Our results identify the miR-34 family as the first functionally important miRNAs for spermatogenesis whose deregulation is causal to oligoasthenoteratozoospermia and infertility.


Assuntos
Astenozoospermia/genética , MicroRNAs/genética , Oligospermia/genética , Animais , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica , Infertilidade Masculina/genética , Masculino , Camundongos Transgênicos , Mitose , Ribonuclease III/genética , Espermatogênese/genética , Espermatozoides/fisiologia
8.
Nat Commun ; 15(1): 6821, 2024 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-39122712

RESUMO

Numerous studies have now demonstrated that lncRNAs can influence gene expression programs leading to cell and organismal phenotypes. Typically, lncRNA perturbations and concomitant changes in gene expression are measured on the timescale of many hours to days. Thus, we currently lack a temporally grounded understanding of the primary, secondary, and tertiary relationships of lncRNA-mediated transcriptional and epigenetic regulation-a prerequisite to elucidating lncRNA mechanisms. To begin to address when and where a lncRNA regulates gene expression, we genetically engineer cell lines to temporally induce the lncRNA Firre. Using this approach, we are able to monitor lncRNA transcriptional regulatory events from 15 min to four days. We observe that upon induction, Firre RNA regulates epigenetic and transcriptional states in trans within 30 min. These early regulatory events result in much larger transcriptional changes after 12 h, well before current studies monitor lncRNA regulation. Moreover, Firre-mediated gene expression changes are epigenetically remembered for days. Overall, this study suggests that lncRNAs can rapidly regulate gene expression by establishing persistent epigenetic and transcriptional states.


Assuntos
Epigênese Genética , RNA Longo não Codificante , Transcrição Gênica , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Humanos , Regulação da Expressão Gênica , Linhagem Celular , Fatores de Tempo
9.
Elife ; 122023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36719724

RESUMO

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation.


Assuntos
RNA Longo não Codificante , Gravidez , Feminino , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transição Epitelial-Mesenquimal , Endoderma , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Diferenciação Celular/genética
10.
iScience ; 24(7): 102762, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34278268

RESUMO

Spermatogonial stem cells (SSCs) sustain spermatogenesis and fertility throughout adult male life. The conserved RNA-binding protein NANOS2 is essential for the maintenance of SSCs, but its targets and mechanisms of function are not fully understood. Here, we generated a fully functional epitope-tagged Nanos2 mouse allele and applied the highly stringent cross-linking and analysis of cDNAs to define NANOS2 RNA occupancy in SSC lines. NANOS2 recognizes the AUKAAWU consensus motif, mostly found in the 3' untranslated region of defined messenger RNAs (mRNAs). We find that NANOS2 is a regulator of key signaling and metabolic pathways whose dosage or activity are known to be critical for SSC maintenance. NANOS2 interacts with components of CCR4-NOT deadenylase complex in SSC lines, and consequently, NANOS2 binding reduces the half-lives of target transcripts. In summary, NANOS2 contributes to SSC maintenance through the regulation of target mRNA stability and key self-renewal pathways.

11.
J Exp Med ; 218(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33156926

RESUMO

The mRNA N6-methyladenosine (m6A) modification has emerged as an essential regulator of normal and malignant hematopoiesis. Inactivation of the m6A mRNA reader YTHDF2, which recognizes m6A-modified transcripts to promote m6A-mRNA degradation, results in hematopoietic stem cell (HSC) expansion and compromises acute myeloid leukemia. Here we investigate the long-term impact of YTHDF2 deletion on HSC maintenance and multilineage hematopoiesis. We demonstrate that Ythdf2-deficient HSCs from young mice fail upon serial transplantation, display increased abundance of multiple m6A-modified inflammation-related transcripts, and chronically activate proinflammatory pathways. Consistent with the detrimental consequences of chronic activation of inflammatory pathways in HSCs, hematopoiesis-specific Ythdf2 deficiency results in a progressive myeloid bias, loss of lymphoid potential, HSC expansion, and failure of aged Ythdf2-deficient HSCs to reconstitute multilineage hematopoiesis. Experimentally induced inflammation increases YTHDF2 expression, and YTHDF2 is required to protect HSCs from this insult. Thus, our study positions YTHDF2 as a repressor of inflammatory pathways in HSCs and highlights the significance of m6A in long-term HSC maintenance.


Assuntos
Adenosina/análogos & derivados , Células-Tronco Hematopoéticas/metabolismo , Inflamação/genética , Proteínas de Ligação a RNA/metabolismo , Adenosina/metabolismo , Animais , Linhagem da Célula , Proliferação de Células , Senescência Celular , Deleção de Genes , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Inflamação/patologia , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Genome Biol ; 21(1): 237, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32894169

RESUMO

BACKGROUND: Several long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function. RESULTS: We previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function. CONCLUSIONS: Our results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.


Assuntos
Fertilidade/genética , RNA Longo não Codificante/genética , Animais , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Fases de Leitura Aberta , Espermatogênese/genética
13.
Cell Res ; 29(3): 221-232, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30617251

RESUMO

Several developmental stages of spermatogenesis are transcriptionally quiescent which presents major challenges associated with the regulation of gene expression. Here we identify that the zygotene to pachytene transition is not only associated with the resumption of transcription but also a wave of programmed mRNA degradation that is essential for meiotic progression. We explored whether terminal uridydyl transferase 4- (TUT4-) or TUT7-mediated 3' mRNA uridylation contributes to this wave of mRNA degradation during pachynema. Indeed, both TUT4 and TUT7 are expressed throughout most of spermatogenesis, however, loss of either TUT4 or TUT7 does not have any major impact upon spermatogenesis. Combined TUT4 and TUT7 (TUT4/7) deficiency results in embryonic growth defects, while conditional gene targeting revealed an essential role for TUT4/7 in pachytene progression. Loss of TUT4/7 results in the reduction of miRNA, piRNA and mRNA 3' uridylation. Although this reduction does not greatly alter miRNA or piRNA expression, TUT4/7-mediated uridylation is required for the clearance of many zygotene-expressed transcripts in pachytene cells. We find that TUT4/7-regulated transcripts in pachytene spermatocytes are characterized by having long 3' UTRs with length-adjusted enrichment for AU-rich elements. We also observed these features in TUT4/7-regulated maternal transcripts whose dosage was recently shown to be essential for sculpting a functional maternal transcriptome and meiosis. Therefore, mRNA 3' uridylation is a critical determinant of both male and female germline transcriptomes. In conclusion, we have identified a novel requirement for 3' uridylation-programmed zygotene mRNA clearance in pachytene spermatocytes that is essential for male meiotic progression.


Assuntos
Prófase Meiótica I/genética , Estágio Paquíteno/genética , Processamento Pós-Transcricional do RNA/fisiologia , Espermatogênese/genética , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estabilidade de RNA/genética , RNA Mensageiro/genética , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo
14.
Cell Stem Cell ; 25(1): 137-148.e6, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31031138

RESUMO

Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion.


Assuntos
Leucemia Mieloide Aguda/terapia , Células-Tronco Neoplásicas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Autorrenovação Celular , Hematopoese , Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Células THP-1
15.
Nat Struct Mol Biol ; 25(9): 778-786, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30104661

RESUMO

RNA viruses are a major threat to animals and plants. RNA interference (RNAi) and the interferon response provide innate antiviral defense against RNA viruses. Here, we performed a large-scale screen using Caenorhabditis elegans and its natural pathogen the Orsay virus (OrV), and we identified cde-1 as important for antiviral defense. CDE-1 is a homolog of the mammalian TUT4 and TUT7 terminal uridylyltransferases (collectively called TUT4(7)); its catalytic activity is required for its antiviral function. CDE-1 uridylates the 3' end of the OrV RNA genome and promotes its degradation in a manner independent of the RNAi pathway. Likewise, TUT4(7) enzymes uridylate influenza A virus (IAV) mRNAs in mammalian cells. Deletion of TUT4(7) leads to increased IAV mRNA and protein levels. Collectively, these data implicate 3'-terminal uridylation of viral RNAs as a conserved antiviral defense mechanism.


Assuntos
Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/virologia , Imunidade Inata , RNA Nucleotidiltransferases/metabolismo , Vírus de RNA/metabolismo , Células A549 , Animais , Caenorhabditis elegans/genética , Humanos , Interferência de RNA , Vírus de RNA/imunologia , Vírus de RNA/fisiologia , RNA Viral/metabolismo , Transcriptoma , Replicação Viral
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