RESUMO
In early embryogenesis of fast cleaving embryos, DNA synthesis is short and surveillance mechanisms preserving genome integrity are inefficient, implying the possible generation of mutations. We have analyzed mutagenesis in Xenopus laevis and Drosophila melanogaster early embryos. We report the occurrence of a high mutation rate in Xenopus and show that it is dependent upon the translesion DNA synthesis (TLS) master regulator Rad18. Unexpectedly, we observed a homology-directed repair contribution of Rad18 in reducing the mutation load. Genetic invalidation of TLS in the pre-blastoderm Drosophila embryo resulted in reduction of both the hatching rate and single-nucleotide variations on pericentromeric heterochromatin in adult flies. Altogether, these findings indicate that during very early Xenopus and Drosophila embryos TLS strongly contributes to the high mutation rate. This may constitute a previously unforeseen source of genetic diversity contributing to the polymorphisms of each individual with implications for genome evolution and species adaptation.
Assuntos
DNA/metabolismo , Drosophila melanogaster , Xenopus laevis , Animais , Reparo do DNA , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Heterocromatina , Mutagênese , Taxa de Mutação , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismoRESUMO
RNA interference (RNAi) requires RNA-dependent RNA polymerases (RdRPs) in many eukaryotes, and RNAi amplification constitutes the only known function for eukaryotic RdRPs. Yet in animals, classical model organisms can elicit RNAi without possessing RdRPs, and only nematode RNAi was shown to require RdRPs. Here we show that RdRP genes are much more common in animals than previously thought, even in insects, where they had been assumed not to exist. RdRP genes were present in the ancestors of numerous clades, and they were subsequently lost at a high frequency. In order to probe the function of RdRPs in a deuterostome (the cephalochordate Branchiostoma lanceolatum), we performed high-throughput analyses of small RNAs from various Branchiostoma developmental stages. Our results show that Branchiostoma RdRPs do not appear to participate in RNAi: we did not detect any candidate small RNA population exhibiting classical siRNA length or sequence features. Our results show that RdRPs have been independently lost in dozens of animal clades, and even in a clade where they have been conserved (cephalochordates) their function in RNAi amplification is not preserved. Such a dramatic functional variability reveals an unexpected plasticity in RNA silencing pathways.
Assuntos
RNA Polimerase Dependente de RNA/genética , Animais , Células Eucarióticas/fisiologia , Anfioxos/genética , Filogenia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/genéticaRESUMO
Assessment of the functionality of individual microRNA/target sites is a crucial issue. Genome editing techniques should theoretically permit a fine functional exploration of such interactions, allowing the mutation of microRNAs or individual binding sites in a complete in vivo setting, therefore abrogating or restoring individual interactions on demand. A major limitation to this experimental strategy is the influence of microRNA sequence on its accumulation level, which introduces a confounding effect when assessing phenotypic rescue by compensatorily mutated microRNA and target site. Here we describe a simple assay to identify microRNA variants most likely to accumulate at wild-type levels even though their sequence has been mutated. In this assay, quantification of a reporter construct in cultured cells predicts the efficiency of an early biogenesis step, the Drosha-dependent cleavage of microRNA precursors, which appears to be a major determinant of microRNA accumulation in our variant collection. This system allowed the generation of a mutant Drosophila strain expressing a bantam microRNA variant at wild-type levels.
Assuntos
MicroRNAs , MicroRNAs/genética , MicroRNAs/metabolismo , Células Cultivadas , Mutação , Sítios de LigaçãoRESUMO
Translational control of maternal mRNA through regulation of poly(A) tail length is crucial during early development. The nuclear poly(A) binding protein, PABP2, was identified biochemically from its role in nuclear polyadenylation. Here, we analyze the in vivo function of PABP2 in Drosophila. PABP2 is required in vivo for polyadenylation, and Pabp2 function, including poly(A) polymerase stimulation, is essential for viability. We also demonstrate an unanticipated cytoplasmic function for PABP2 during early development. In contrast to its role in nuclear polyadenylation, cytoplasmic PABP2 acts to shorten the poly(A) tails of specific mRNAs. PABP2, together with the deadenylase CCR4, regulates the poly(A) tails of oskar and cyclin B mRNAs, both of which are also controlled by cytoplasmic polyadenylation. Both Cyclin B protein levels and embryonic development depend upon this regulation. These results identify a regulator of maternal mRNA poly(A) tail length and highlight the importance of this mode of translational control.
Assuntos
Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína II de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Padronização Corporal , Ciclo Celular/fisiologia , Ciclina B/genética , Ciclina B/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Feminino , Masculino , Dados de Sequência Molecular , Oócitos/fisiologia , Proteína II de Ligação a Poli(A)/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismoRESUMO
I elements in Drosophila melanogaster are non-long terminal repeat (LTR) retrotransposons of particular interest because high levels of transposition can be induced by appropriate crosses. They use a full-length RNA transposition intermediate as a template for reverse transcription. Detailed molecular characterization of this intermediate is rendered difficult because of the many transcripts produced by defective elements. The use of an active I element marked with a sequence encoding the HA epitope solves this problem. We used an RNA circularization procedure followed by RT-PCR to analyze the transcripts produced by actively transposing tagged I elements. Most start at the 5' end at the second nucleotide of the I element and all are polyadenylated at a site located in genomic sequences downstream of the 3' end. One of the tagged I elements, inserted in locus 88A, produces chimeric transcripts that carry sequences from both 5'- and 3'-flanking genomic DNA. We show that synthesis of these chimeric transcripts is controlled by the I element itself. Analysis of full-length transposed copies of this element shows that the extra sequences at the 5' and 3' ends are not integrated during retrotransposition. This suggests that initiation and arrest of reverse transcription during retrotransposition are precise processes.
Assuntos
Drosophila melanogaster/genética , RNA/genética , Retroelementos , Transcrição Gênica , Região 3'-Flanqueadora , Região 5'-Flanqueadora , Animais , Animais Geneticamente Modificados , Sequência de Bases , Feminino , Hemaglutininas/genética , Dados de Sequência Molecular , Poliadenilação , RNA/biossíntese , RNA/química , Capuzes de RNA/química , RNA Circular , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Several studies have recently shown that the activity of some eukaryotic transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent gene-silencing mechanisms in their regulation. Here we provide data indicating that two non-LTR retrotransposons of Drosophila melanogaster are themselves natural triggers of homology-dependent gene silencing. We show that, in the female germline of D. melanogaster, fragments from the R1 or from the I retrotransposons can mediate silencing of chimeric transcription units into which they are inserted. This silencing is probably mediated by sequence identity with endogenous copies of the retrotransposons because it does not occur with a fragment from the divergent R1 elements of Bombyx mori, and, when a fragment of I is used, it occurs only in females containing functional copies of the I element. This silencing is not accompanied by cosuppression of the endogenous gene homologous to the chimeric transcription unit, which contrasts to some other silencing mechanisms in Drosophila. These observations suggest that in the female germline of D. melanogaster the R1 and I retrotransposons may self-regulate their own activity and their copy number by triggering homology-dependent gene silencing.
Assuntos
Drosophila melanogaster/genética , Inativação Gênica , Retroelementos/genética , Sequências Repetidas Terminais/genética , Animais , Animais Geneticamente Modificados , Bombyx/genética , Cruzamentos Genéticos , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Feminino , Hibridização In Situ , Masculino , Modelos Genéticos , Proteínas Nucleares/genética , Ovário/metabolismo , Plasmídeos , TransgenesRESUMO
Drosophila Orb, the homolog of vertebrate CPEB, is a key translational regulator involved in oocyte polarity and maturation through poly(A) tail elongation of specific mRNAs. orb also has an essential function during early oogenesis that has not been addressed at the molecular level. Here, we show that orb prevents cell death during early oogenesis, thus allowing oogenesis to progress. It does so through the repression of autophagy by directly repressing, together with the CCR4 deadenylase, the translation of Autophagy-specific gene 12 (Atg12) mRNA. Autophagy and cell death observed in orb mutant ovaries are reduced by decreasing Atg12 or other Atg mRNA levels. These results reveal a role of Orb in translational repression and identify autophagy as an essential pathway regulated by Orb during early oogenesis. Importantly, they also establish translational regulation as a major mode of control of autophagy, a key process in cell homeostasis in response to environmental cues.
Assuntos
Autofagia/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Ciclo Celular , Morte Celular , Drosophila/metabolismo , Feminino , Células Germinativas/metabolismo , Homeostase , Imunoprecipitação , Mutação , Oócitos/metabolismo , Oogênese , Ovário/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleases/metabolismoRESUMO
The current model of short interspersed nuclear element (SINE) mobility suggests that these non-coding retroposons are able to recruit for their own benefits the enzymatic machinery encoded by autonomous long interspersed nuclear elements (LINEs). The recent characterization of potential SINE-LINE partner pairs that share common 3' end sequences concurs with this model and has led to a potent picture of tRNA-derived SINEs consisting of a tripartite functional structure (Mol. Cell. Biol. 16 (1996) 3756; Mol. Biol. Evol. 16 (1999) 1238; Proc. Natl. Acad. Sci. USA 96 (1999) 2869). This structure consist of a 5' polIII tRNA-related promoter region, a central conserved domain and a variable 3' region with homology to the 3' end of LINEs, believed to be essential to direct recognition by the LINE proteins. To test this model in vivo, we have designed synthetic SINEs possessing this 'canonical' structure, including 3' homology to the 3' UTR of the LINE I factor from Drosophila. These synthetic elements were introduced in a Drosophila reactive strain, and SINE retroposition was assessed following dysgenic crosses that are known to induce high levels of I factor germinal transposition. In the progeny from the dysgenic crosses 3400-4000 flies were analyzed but no retroposed copy of the chimeric SINEs was detected, indicating that what is assumed to be a typical SINE structure is not sufficient per se to allow efficient trans-mobilization of our synthetic SINEs by an actively amplifying partner LINE. Alternatively, the apparent absence of natural fly SINEs may underline intrinsic properties of fly biology that are incompatible with the genesis and/or propagation of SINE-like elements.
Assuntos
Drosophila melanogaster/genética , Retroelementos/genética , Elementos Nucleotídeos Curtos e Dispersos/genética , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Feminino , Expressão Gênica , Masculino , Mutagênese Insercional , Plasmídeos/genética , RNA/genética , RNA/metabolismoRESUMO
Translational regulation plays an essential role in Drosophila ovarian germline stem cell (GSC) biology. GSC self-renewal requires two translational repressors, Nanos (Nos) and Pumilio (Pum), which repress the expression of differentiation factors in the stem cells. The molecular mechanisms underlying this translational repression remain unknown. Here, we show that the CCR4 deadenylase is required for GSC self-renewal and that Nos and Pum act through its recruitment onto specific mRNAs. We identify mei-P26 mRNA as a direct and major target of Nos/Pum/CCR4 translational repression in the GSCs. mei-P26 encodes a protein of the Trim-NHL tumor suppressor family that has conserved functions in stem cell lineages. We show that fine-tuning Mei-P26 expression by CCR4 plays a key role in GSC self-renewal. These results identify the molecular mechanism of Nos/Pum function in GSC self-renewal and reveal the role of CCR4-NOT-mediated deadenylation in regulating the balance between GSC self-renewal and differentiation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Óvulo/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/metabolismo , Células-Tronco/metabolismo , Animais , Linhagem da Célula , Proliferação de Células , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Feminino , Oogênese , Óvulo/citologia , Óvulo/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleases/genética , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Anteroposterior patterning of the Drosophila embryo depends on a gradient of Nanos protein arising from the posterior pole. This gradient results from both nanos mRNA translational repression in the bulk of the embryo and translational activation of nanos mRNA localized at the posterior pole. Two mechanisms of nanos translational repression have been described, at the initiation step and after this step. Here we identify a novel level of nanos translational control. We show that the Smaug protein bound to the nanos 3' UTR recruits the deadenylation complex CCR4-NOT, leading to rapid deadenylation and subsequent decay of nanos mRNA. Inhibition of deadenylation causes stabilization of nanos mRNA, ectopic synthesis of Nanos protein and head defects. Therefore, deadenylation is essential for both translational repression and decay of nanos mRNA. We further propose a mechanism for translational activation at the posterior pole. Translation of nanos mRNA at the posterior pole depends on oskar function. We show that Oskar prevents the rapid deadenylation of nanos mRNA by precluding its binding to Smaug, thus leading to its stabilization and translation. This study provides insights into molecular mechanisms of regulated deadenylation by specific proteins and demonstrates its importance in development.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Western Blotting , Primers do DNA , Drosophila/metabolismo , Imunoprecipitação , Hibridização In Situ , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismoRESUMO
Non-long terminal repeat retrotransposons, widespread among eukaryotic genomes, transpose by reverse transcription of an RNA intermediate. Some of them, like L1 in the human, terminate at the 3'-end with a poly(dA) stretch whereas others, like the I factor in Drosophila melanogaster, have instead a short sequence repeated in tandem. This suggests different requirements for the initiation of reverse transcription. Here, we have used an RNA circularization/reverse transcription-PCR technique to analyze the 5'- and 3'-ends of the full-length transcripts produced by the I factor at the time of active retrotransposition. These transcripts are capped and polyadenylated similar to conventional messenger RNAs. We have analyzed the 3'-ends of transcripts and transposed copies produced by I elements mutated at the 3'-ends. Transcripts devoid of tandem UAA repeats, although capable of building the components of the retrotransposition machinery, are inefficiently used as retrotransposition intermediates. Such transcripts produce rare new integrated copies issued from the inaccurate initiation of reverse transcription near the 3'-end of the element. The tandem UAA repeats at the 3'-end of the transcripts of I are required for the efficient and precise initiation of reverse transcription. This strong specificity of the I factor reverse transcriptase for its own transcript has implications for the impact of I factor retrotransposition on the host genome.