RESUMEN

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and is caused by mutations in the gene encoding the Fragile X messenger ribonucleoprotein (FMRP). FMRP is an evolutionarily conserved and neuronally enriched RNA-binding protein (RBP) with functions in RNA editing, RNA transport, and protein translation. Specific target RNAs play critical roles in neurodevelopment, including the regulation of neurite morphogenesis, synaptic plasticity, and cognitive function. The different biological functions of FMRP are modulated by its cooperative interaction with distinct sets of neuronal RNA and protein-binding partners. Here, we focus on interactions between FMRP and components of the microRNA (miRNA) pathway. Using the Drosophila S2 cell model system, we show that the Drosophila ortholog of FMRP (dFMRP) can repress translation when directly tethered to a reporter mRNA. This repression requires the activity of AGO1, GW182, and MOV10/Armitage, conserved proteins associated with the miRNA-containing RNA-induced silencing complex (miRISC). Additionally, we find that untagged dFMRP can interact with a short stem-loop sequence in the translational reporter, a prerequisite for repression by exogenous miR-958. Finally, we demonstrate that dFmr1 interacts genetically with GW182 to control neurite morphogenesis. These data suggest that dFMRP may recruit the miRISC to nearby miRNA binding sites and repress translation via its cooperative interactions with evolutionarily conserved components of the miRNA pathway.

Asunto(s)

Proteínas de Drosophila , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , MicroARNs , Neuritas , Biosíntesis de Proteínas , Animales , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , MicroARNs/genética , MicroARNs/metabolismo , Neuritas/metabolismo , Morfogénesis/genética , Complejo Silenciador Inducido por ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Drosophila/metabolismo , Drosophila/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Unión Proteica

RESUMEN

MicroRNAs (miRNAs) are a class of small regulatory RNA that are generated via core protein machinery. The miRNAs direct gene-silencing mechanisms to mediate an essential role in gene expression regulation. In mollusks, miRNAs have been demonstrated to be required to regulate gene expression in various biological processes, including normal development, immune responses, reproduction, and stress adaptation. In this study, we aimed to establishment the requirement of the miRNA pathway as part of the molecular response of exposure of Biomphalaria glabrata (snail host) to Schistosoma mansoni (trematode parasite). Initially, the core pieces of miRNA pathway protein machinery, i.e., Drosha, DGCR8, Exportin-5, Ran, and Dicer, together with the central RNA-induced silencing complex (RISC) effector protein Argonaute2 (Ago2) were elucidated from the B. glabrata genome. Following exposure of B. glabrata to S. mansoni miracidia, we identified significant expression up-regulation of all identified pieces of miRNA pathway protein machinery, except for Exportin-5, at 16 h post exposure. For Ago2, we went on to show that the Bgl-Ago2 protein was localized to regions surrounding the sporocysts in the digestive gland of infected snails 20 days post parasite exposure. In addition to documenting elevated miRNA pathway protein machinery expression at the early post-exposure time point, a total of 13 known B. glabrata miRNAs were significantly differentially expressed. Of these thirteen B. glabrata miRNAs responsive to S. mansoni miracidia exposure, five were significantly reduced in their abundance, and correspondingly, these five miRNAs were determined to putatively target six genes with significantly elevated expression and that have been previously associated with immune responses in other animal species, including humans. In conclusion, this study demonstrates the central importance of a functional miRNA pathway in snails, which potentially forms a critical component of the immune response of snails to parasite exposure. Further, the data reported in this study provide additional evidence of the complexity of the molecular response of B. glabrata to S. mansoni infection: a molecular response that could be targeted in the future to overcome parasite infection and, in turn, human schistosomiasis.

Asunto(s)

Proteínas Argonautas , Biomphalaria , MicroARNs , Schistosoma mansoni , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Biomphalaria/parasitología , Biomphalaria/genética , Biomphalaria/metabolismo , Biomphalaria/inmunología , MicroARNs/genética , MicroARNs/metabolismo , Schistosoma mansoni/genética , Oocistos/metabolismo , Interacciones Huésped-Parásitos/genética

RESUMEN

Germ cells mutant for bam or bgcn are locked in a germline stem cell (GSC)-like state, leading to tumor-like overgrowth in Drosophila ovaries. Our previous studies have demonstrated that germline overgrowth in bam mutants can be suppressed by defects in the miRNA pathway but enhanced by a null mutation in hippo. However, the genetic epistasis between the miRNA and Hippo pathways still remains unknown. Here, we determined that the miRNA pathway acts downstream of the Hippo pathway in regulating this process. Germ cells mutant for bam or bgcn and defective in both pathways divide very slowly, phenocopying those defective only in the miRNA pathway. In addition, we found that Yki, a key oncoprotein in the Hippo pathway, promotes the growth of both wild-type germ cells and bam mutant GSC-like cells. Like wild-type GSCs, bam mutant GSC-like cells predominantly stay in the G2 phase. Remarkably, many of those defective in the miRNA pathway are arrested before entering this phase. Furthermore, our studies identified bantam as a critical miRNA promoting germline overgrowth in bam or bgcn mutants. Taken together, these findings establish a genetic circuitry controlling Drosophila female germline overgrowth.

Asunto(s)

Proteínas de Drosophila , Células Germinativas , MicroARNs , Ovario , Proteínas Señalizadoras YAP , Animales , Femenino , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Células Germinativas/metabolismo , Ovario/metabolismo , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transducción de Señal/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Mutación/genética , Transactivadores/genética , Transactivadores/metabolismo , ADN Helicasas

RESUMEN

DNA methylation is an epigenetic process that involves the chemical modification of DNA, leading to the regulation of its transcriptional activity. It is primarily known for the addition of methyl groups to cytosine in DNA. The whitefly Bemisia tabaci is a polyphagous pest insect and a vector that is responsible for transmitting numerous plant viruses, resulting in significant economic losses in agricultural crops globally. In our study, we characterized the expression of two key DNA methylation genes, the DNA methyltransferases Dnmt1 and Dnmt3, in B. tabaci. Additionally, we explored the impact of inhibiting DNMTs on the miRNA pathway and fitness of whitefly. To investigate the role of the DNA methylation pathway in B. tabaci, we found that the expression of Dnmt1 and Dnmt3 varied across different tissues and developmental stages of B. tabaci. We employed azacytidine (5-AZA) treatment of adults to inhibit DNMTs (DNMT1 and DNMT3). Administration of 5-AZA affected the survival and reproduction of this pest. Moreover, inhibition of DNMTs led to a decrease in the expression of the miRNA pathway core genes Dicer1 and Argonaute1, which subsequently resulted in reduced expression of Let-7 and miR-184 which are essential microRNAs in the physiology and biology of insects. The study suggests that DNA methyltransferases could be targeted for developing an inhibition strategy to control this pest and vector insect.

Asunto(s)

Metilación de ADN , Hemípteros , MicroARNs , Animales , Hemípteros/genética , MicroARNs/genética , MicroARNs/metabolismo , Azacitidina/farmacología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Femenino

RESUMEN

MicroRNAs (miRNAs) are noncoding small regulatory RNAs involved in diverse biological processes. Odontotermes formosanus (Shiraki) is a polyphagous pest that causes economic damage to agroforestry. Serratia marcescens is a bacterium with great potential for controlling this insect. However, knowledge about the miRNA pathway and the role of miRNAs in O. formosanus defense against SM1 is limited. In this study, OfAgo1, OfDicer1 and OfDrosha were differentially expressed in different castes and tissues. SM1 infection affected the expression of all three genes in O. formosanus. Then, we used specific double-stranded RNAs to silence OfAgo1, OfDicer1 and OfDrosha. Knockdown of these genes enhanced the virulence of SM1 to O. formosanus, suggesting that miRNAs were critical in the defense of O. formosanus against SM1. Furthermore, we sequenced miRNAs from SM1-infected and uninfected O. formosanus. 33 differentially expressed (DE) miRNAs were identified, whereby 22 were upregulated and 11 were downregulated. Finally, the miRNA-mRNA networks were constructed, which further suggested the important role of miRNAs in the defense of O. formosanus against SM1. Totally, O. formosanus miRNA core genes defend against SM1 infection by regulating miRNA expression. This study elucidates the interactions between O. formosanus and SM1 and provides new theories for biological control.

Asunto(s)

MicroARNs , Serratia marcescens , MicroARNs/genética , MicroARNs/metabolismo , Serratia marcescens/genética , Serratia marcescens/patogenicidad , Animales , Escarabajos/microbiología , Escarabajos/genética

RESUMEN

We applied a new RNA interference (RNAi) system using rolling circle transcription (RCT) technology to generate RNA microspheres (RMS) for targeting two key chitin synthetic pathway genes [chitin synthase A (CHSA), chitin synthase B (CHSB)] in the larvae of the oriental armyworm (Mythimna separate), a RNAi-unsusceptible agriculturally important lepidopteran pest. Feeding the third-instar larvae with the RMS-CHSA- or RMS-CHSB-treated corn leaf discs suppressed the expression of CHSA by 81.7% or CHSB by 88.1%, respectively, at 72 h. The silencing of CHSA consequently affected the larval development, including the reduced body weight (54.0%) and length (41.3%), as evaluated on the 7th day, and caused significant larval mortalities (51.1%) as evaluated on the 14th day. Similar results were obtained with the larvae fed RMS-CHSB. We also compared RNAi efficiencies among different strategies: 1) two multi-target RMS [i.e., RMS-(CHSA + CHSB), RMS-CHSA + RMS-CHSB], and 2) multi-target RMS and single-target RMS (i.e., either RMS-CHSA or RMS-CHSB) and found no significant differences in RNAi efficiency. By using Cy3-labeled RMS, we confirmed that RMS can be rapidly internalized into Sf9 cells (<6 h). The rapid cellular uptake of RMS accompanied with significant RNAi efficiency through larval feeding suggests that the RCT-based RNAi system can be readily applied to study the gene functions and further developed as bio-pesticides for insect pest management. Additionally, our new RNAi system takes the advantage of the microRNA (miRNA)-mediated RNAi pathway using miRNA duplexes generated in vivo from the RMS by the target insect. The system can be used for RNAi in a wide range of insect species, including lepidopteran insects which often exhibit extremely low RNAi efficiency using other RNAi approaches.

Asunto(s)

MicroARNs , Mariposas Nocturnas , Animales , Interferencia de ARN , Quitina Sintasa/genética , Quitina Sintasa/metabolismo , Microesferas , Mariposas Nocturnas/genética , Mariposas Nocturnas/metabolismo , Insectos/genética , Larva/metabolismo , ARN Bicatenario

RESUMEN

RNA interference (RNAi) is a specific post-transcriptional gene-silencing phenomenon, which plays an important role in the regulation of gene expression and the protection from transposable elements in eukaryotic organisms. In Drosophila melanogaster, RNAi can be induced by microRNA (miRNA), endogenous small interfering RNA (siRNA), or exogenous siRNA. However, the biogenesis of miRNA and siRNA in these RNAi pathways is aided by the double-stranded RNA binding proteins (dsRBPs) Loquacious (Loqs)-PB, Loqs-PD or R2D2. In this study, we identified three alternative splicing variants of Loqs, namely Loqs-PA, -PB, and -PC in the orthopteran Locusta migratoria. We performed in vitro and in vivo experiments to study the roles of the three Loqs variants in the miRNA- and siRNA-mediated RNAi pathways. Our results show that Loqs-PB assists the binding of pre-miRNA to Dicer-1 to lead to the cleavage of pre-miRNA to yield matured miRNA in the miRNA-mediated RNAi pathway. In contrast, different Loqs proteins participate in different siRNA-mediated RNAi pathways. In exogenous siRNA-mediated RNAi pathway, binding of Loqs-PA or LmLoqs-PB to exogenous dsRNA facilitates the cleavage of dsRNA by Dicer-2, whereas in endogenous siRNA-mediated RNAi pathway, binding of Loqs-PB or Loqs-PC to endogenous dsRNA facilitates the cleavage of dsRNA by Dicer-2. Our findings provide new insights into the functional importance of different Loqs proteins derived from alternative splicing variants of Loqs in achieving high RNAi efficiency in different RNAi pathways in insects.

Asunto(s)

Empalme Alternativo , Locusta migratoria , MicroARNs , ARN Interferente Pequeño , Animales , Locusta migratoria/genética , MicroARNs/genética , Interferencia de ARN , ARN Bicatenario/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN

RESUMEN

MicroRNAs (miRNAs) are small single-stranded non-coding RNAs involved in a variety of cellular events by regulating gene expression at the post-transcriptional level. Several core genes in miRNA biogenesis have been reported to participate in a wide range of physiological events, in some insect species. However, the functional significance of miRNA pathway core genes in Nilaparvata lugens remains unknown. In the present study, we conducted a systematic characterisation of five core genes involved in miRNA biogenesis. We first performed spatiotemporal expression analysis and found that miRNA core genes exhibited similar expression patterns, with high expression levels in eggs and relatively high transcriptional levels in the ovaries and fat bodies of females. RNA interference experiments showed that injecting third-instar nymphs with dsRNAs targeting the miRNA core genes, NlAgo1, NlDicer1, and NlDrosha resulted in high mortality rates and various degrees of body melanism, moulting defects, and wing deformities. Further investigations revealed that the suppression of miRNA core genes severely impaired ovarian development and oocyte maturation, resulting in significantly reduced fecundity and disruption of intercellular spaces between follicle cells. Moreover, the expression profiles of miR-34-5p, miR-275-3p, miR-317-3p, miR-14, Let-7-1, and miR-2a-3p were significantly altered in response to the knockdown of miRNA core genes mixture, suggesting that they play essential roles in regulating miRNA-mediated gene expression. Therefore, our results provide a solid theoretical basis for the miRNA pathway in N. lugens and suggest that the NlAgo1, NlDicer1, and NlDrosha-dependent miRNA core genes are essential for the development and reproduction of this agricultural pest.

Asunto(s)

Hemípteros , MicroARNs , Femenino , Animales , Reproducción , Interferencia de ARN , MicroARNs/genética , MicroARNs/metabolismo , Fertilidad/genética , Hemípteros/fisiología

RESUMEN

BACKGROUND: MicroRNAs (miRNAs) are small regulatory non-coding RNAs that are involved in a variety of biological processes such as immunity, cell signaling and development by regulating gene expression. The whitefly Bemisia tabaci is a polyphagous vector that transmits many plant viruses causing economic damage to crops worldwide. In this study, we characterized and analyzed the expression of the miRNA core genes Argonaute-1 (Ago1) and Dicer-1 (Dcr1) in B. tabaci and explored the effect of their silencing on the insect's fitness. RESULTS: Our results showed that Ago1 and Dcr1 are differentially expressed in different tissues and developmental stages of B. tabaci. To determine the function of the miRNA pathway in B. tabaci, we silenced Ago1 and Dcr1 using specific double-stranded RNAs to the genes. RNA interference (RNAi) of Ago1 and Dcr1 decreased the expression level of the core genes and reduced the abundance of Let-7 and miR-184 miRNAs. Silencing of the miRNA pathway core gene also negatively affected the biology of B. tabaci by reducing fertility, fecundity and survival of this insect pest. CONCLUSIONS: Together, our results showed that silencing the miRNA pathway core genes reduced the miRNA levels followed by reduced fecundity and survival of B. tabaci, which highlighted the importance of the miRNA pathway in this insect. The miRNA core genes are attractive targets for developing an RNAi-based strategy for targeting this notorious insect pest. © 2022 Society of Chemical Industry.

Asunto(s)

Hemípteros , MicroARNs , Animales , Fertilidad/genética , Hemípteros/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Interferencia de ARN , ARN Bicatenario

RESUMEN

The stem cell-specific RNA-binding protein TRIM71/LIN-41 was the first identified target of the pro-differentiation and tumor suppressor miRNA let-7. TRIM71 has essential functions in embryonic development and a proposed oncogenic role in several cancer types, such as hepatocellular carcinoma. Here, we show that TRIM71 regulates let-7 expression and activity via two independent mechanisms. On the one hand, TRIM71 enhances pre-let-7 degradation through its direct interaction with LIN28 and TUT4, thereby inhibiting let-7 maturation and indirectly promoting the stabilization of let-7 targets. On the other hand, TRIM71 represses the activity of mature let-7 via its RNA-dependent interaction with the RNA-Induced Silencing Complex (RISC) effector protein AGO2. We found that TRIM71 directly binds and stabilizes let-7 targets, suggesting that let-7 activity inhibition occurs on active RISCs. MiRNA enrichment analysis of several transcriptomic datasets from mouse embryonic stem cells and human hepatocellular carcinoma cells suggests that these let-7 regulatory mechanisms shape transcriptomic changes during developmental and oncogenic processes. Altogether, our work reveals a novel role for TRIM71 as a miRNA repressor and sheds light on a dual mechanism of let-7 regulation.

RESUMEN

Varroa destructor is an ectoparasite mite that attacks bees leading to colony disorders worldwide. microRNAs (miRNAs) are key molecules used by eukaryotes to post-transcriptional control of gene expression. Nevertheless, still lack information aboutV. destructor miRNAs and its regulatory networks. Here, we used an integrative strategy to characterize the miRNAs in the V. destructor mite. We identified 310 precursors that give rise to 500 mature miRNAs, which 257 are likely mite-specific elements. miRNAs showed canonical length ranging between 18 and 25 nucleotides and 5' uracil preference. Top 10 elements concentrated over 80% of total miRNA expression, with bantam alone representing ~50%. We also detected non-templated bases in precursor-derived small RNAs, indicative of miRNA post-transcriptional regulatory mechanisms. Finally, we note that conserved miRNAs control similar processes in different organisms, suggesting a conservative role. Altogether, our findings contribute to the better understanding of the mite biology that can assist future studies on varroosis control.

Asunto(s)

MicroARNs , Varroidae , Animales , Abejas/parasitología , Regulación de la Expresión Génica , Genoma , MicroARNs/genética , Varroidae/genética

RESUMEN

The pea aphid is an important pest of vegetables and causes serious losses worldwide. RNA interference (RNAi) is an effective pest control tool, and three sub-pathways have been described: The miRNA pathway, siRNA pathway, and piRNA pathway. A large number of genes in miRNA pathway and piRNA pathway are found to be expanded. To study the roles of these genes, the expression of 25 core RNAi genes was screened in spatiotemporal samples, artificially synthesized dsRNA and miRNA treated samples. The 25 genes were all expressed during different development stages and in different tissues. In dsRNA-treated samples and miRNA-treated samples, the expressions of genes in these three pathways were induced, especially the expanded genes. This suggests a complex network of RNAi core genes in the three sub-pathways. Treatment of miRNA seems to induce gene expression in a dosage-dependent manner. These results increase our knowledge of the siRNA pathway and related factors from RNAi pathway in aphids and promote the use of RNAi for the control of aphid pests.

RESUMEN

P-bodies are cytoplasmic foci composed of mRNAs and enzymes involved in mRNA degradation. P-bodies have been found to link to RNA interference and RNA decay mediated by microRNAs (miRNAs) and translational repression. Here, we aim to investigate different effects of overexpressed Dcp1a or GW182 on cytoplasmic aggregates formation and influence on miRNA pathway. Small RNAs were recruited into endogenous foci of P-bodies and aggregates formed by Dcpa1 and GW182 overexpression. However, only overexpressed Dcp1a but not GW182 was colocalized with DDX6, another component of P-bodies and suppressed protein translation. In addition, we investigated the relationship between stress granules and miRNA pathway and found that granules induced by G3BP1 overexpression could recruit small RNAs into the granules and repressed protein translation. As Ago2 is a key component of RNA-induced silencing complex, we also investigated the localization of endogenous Ago2 (edo-Ago2) after Dcp1a and GW182 overexpression, and found that endo-Ago2 did not colocalize with the aggregates induced by overexpression of Dcpla, GW182, and G3BP1. Notably, the ability of miRNA to regulate its target was enhanced by the granules induced by Dcp1a and G3BP1 expression. Our results suggest that overexpressed Dcp1a and GW182 can form different cytoplasmic aggregates and play distinct biological roles in the miRNA pathway.

Asunto(s)

Autoantígenos/genética , Proteínas Portadoras/genética , Endorribonucleasas/genética , MicroARNs/genética , Interferencia de ARN , Proteínas de Unión al ARN/genética , Transactivadores/genética , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Autoantígenos/metabolismo , Proteínas Portadoras/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas , Endorribonucleasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , MicroARNs/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Unión Proteica , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Transactivadores/metabolismo

RESUMEN

RNA interference (RNAi), including microRNAs, is an important player in the mediation of differentiation and migration of stem cells via target genes. It is used as a potential strategy for gene therapy for central nervous system (CNS) diseases. Stem cells are considered vectors of RNAi due to their capacity to deliver RNAi to other cells. In this review, we discuss the recent advances in studies of RNAi pathways in controlling neuronal differentiation and migration of stem cells. We also highlight the utilization of a combination of RNAi and stem cells in treatment of CNS diseases.

RESUMEN

Spaced synaptic depolarization induces rapid axon terminal growth and the formation of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). Here, we identify a novel presynaptic function for the Calcium/Calmodulin-dependent Kinase II (CamKII) protein in the control of activity-dependent synaptic growth. Consistent with this function, we find that both total and phosphorylated CamKII (p-CamKII) are enriched in axon terminals. Interestingly, p-CamKII appears to be enriched at the presynaptic axon terminal membrane. Moreover, levels of total CamKII protein within presynaptic boutons globally increase within one hour following stimulation. These effects correlate with the activity-dependent formation of new presynaptic boutons. The increase in presynaptic CamKII levels is inhibited by treatment with cyclohexamide suggesting a protein-synthesis dependent mechanism. We have previously found that acute spaced stimulation rapidly downregulates levels of neuronal microRNAs (miRNAs) that are required for the control of activity-dependent axon terminal growth at this synapse. The rapid activity-dependent accumulation of CamKII protein within axon terminals is inhibited by overexpression of activity-regulated miR-289 in motor neurons. Experiments in vitro using a CamKII translational reporter show that miR-289 can directly repress the translation of CamKII via a sequence motif found within the CamKII 3' untranslated region (UTR). Collectively, our studies support the idea that presynaptic CamKII acts downstream of synaptic stimulation and the miRNA pathway to control rapid activity-dependent changes in synapse structure.

Asunto(s)

Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Potenciales de la Membrana , Proyección Neuronal , Terminales Presinápticos/metabolismo , Regiones no Traducidas 3' , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Línea Celular , Drosophila , MicroARNs/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Terminales Presinápticos/fisiología

RESUMEN

The Drosophila larval ovary morphogenesis mainly involves coordinated development of somatic and germ cell lineages that is essential for forming a correct number of niche-germline stem cell (GSC) units (ovarioles) in the adult ovary. Ecdysone, Insulin, Activin, Dpp and EGFR signaling pathways form a regulatory network that orchestrates ovarian soma and germ line throughout larval development. Identification and characterization of additional genes or machineries involved in this process will provide more insights into the underlying mechanisms. Here, we show that the core microRNA (miRNA) pathway components Drosha and Pasha are required for coordinated development of somatic and germ cell precursors in the larval ovary. Drosha or pasha mutants display defective proliferation of primordial germ cells (PGCs), the precursors of GSCs prior to late third larval instar (LL3) and promoted PGC differentiation at LL3. In the mean time, loss of Drosha or Pasha function perturbs somatic precursor development, causing defects in formation of terminal filaments (TFs), a major composition of the GSC niche at LL3, as well as in TF precursor accumulation at early larval stages. Comparative analysis of the mutant phenotypes reveals that three other key miRNA pathway components, Dicer-1 (Dcr-1), Loquacious (Loqs) and Argonaute-1 (Ago-1) have similar effects as Drosha and Pasha indicated above, suggesting a role of the canonical miRNA pathway in the ovary development. Furthermore, genome-wide screening and genetic studies identify a set of Drosha-controlled miRNAs including miR-8, miR-14, miR-33, miR-184, miR-317 and let-7-C that function in this gonadogenesis. Taken together, this study provides the first ever demonstration that miRNA-mediated regulation is involved in the Drosophila larval ovary morphogenesis.

Asunto(s)

Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , MicroARNs/genética , Ovario/crecimiento & desarrollo , Proteínas de Unión al ARN/fisiología , Ribonucleasa III/fisiología , Animales , Diferenciación Celular , Citoesqueleto/ultraestructura , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Células Germinales Embrionarias/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Larva , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Organogénesis , Ovario/citología , Interferencia de ARN , Proteínas de Unión al ARN/genética , Ribonucleasa III/deficiencia , Ribonucleasa III/genética , Nicho de Células Madre

RESUMEN

The discovery of RNA interference (RNAi) as an endogenous mechanism of gene regulation in a range of eukaryotes has resulted in its extensive use as a tool for functional genomic studies. It is important to study the mechanisms which underlie this phenomenon in different organisms, and in particular to understand details of the effectors that modulate its effectiveness. The aim of this study was to identify and compare genomic sequences encoding genes involved in the RNAi pathway of four parasitic nematodes: the plant parasites Meloidogyne hapla and Meloidogyne incognita and the animal parasites Ascaris suum and Brugia malayi because full genomic sequences were available-in relation to those of the model nematode Caenorhabditis elegans. The data generated was then used to identify some potential targets for control of the root knot nematode, M. incognita. Of the 84 RNAi pathway genes of C. elegans used as model in this study, there was a 42-53 % reduction in the number of effectors in the parasitic nematodes indicating substantial differences in the pathway between species. A gene each from six functional groups of the RNAi pathway of M. incognita was downregulated using in vitro RNAi, and depending on the gene (drh-3, tsn-1, rrf-1, xrn-2, mut-2 and alg-1), subsequent plant infection was reduced by up to 44 % and knockdown of some genes (i.e. drh-3, mut-2) also resulted in abnormal nematode development. The information generated here will contribute to defining targets for more robust nematode control using the RNAi technology.

Asunto(s)

Redes y Vías Metabólicas/genética , Interferencia de ARN , ARN/genética , Tylenchoidea/genética , Animales , Ascaris suum/genética , Ascaris suum/patogenicidad , Brugia Malayi/genética , Brugia Malayi/patogenicidad , Caenorhabditis elegans/genética , Resistencia a la Enfermedad/genética , Técnicas de Silenciamiento del Gen , Genoma , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/parasitología , ARN/biosíntesis , Tylenchoidea/patogenicidad

RESUMEN

Decades of studies have shown evolutionarily conserved molecular networks consisting of transcriptional factors, diffusing growth factors, and signaling pathways that regulate proper lung development. Recently, microRNAs (miRNAs), small, noncoding regulatory RNAs, have been integrated into these networks. Significant advances have been made in characterizing the developmental stage- or cell type-specific miRNAs during lung development by using approaches such as genome-wide profiling and in situ hybridization. Results from gain- or loss-of-function studies revealed pivotal roles of protein components of the miRNA pathway and individual miRNAs in regulating proliferation, apoptosis, differentiation, and morphogenesis during lung development. Aberrant expression or functions of these components have been associated with pulmonary disorders, suggesting their involvement in pathogenesis of these diseases. Moreover, genetically modified mice generated in these studies have become useful models of human lung diseases. Challenges in this field include characterization of collective function and responsible targets of miRNAs specifically expressed during lung development, and translation of these basic findings into clinically relevant information for better understanding of human diseases. The goal of this review is to discuss the recent progress on the understanding of how the miRNA pathway regulates lung development, how dysregulation of miRNA activities contributes to pathogenesis of related pulmonary diseases, and to identify relevant questions and future directions.

Asunto(s)

Enfermedades Pulmonares/metabolismo , MicroARNs/fisiología , Animales , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Pulmón/irrigación sanguínea , Pulmón/embriología , Pulmón/metabolismo , Pulmón/patología , Enfermedades Pulmonares/genética , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/fisiología , Interferencia de ARN , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología