RESUMO
Axon and dendrite development require the cooperation of actin and microtubule cytoskeletons. Microtubules form a well-organized network to direct polarized trafficking and support neuronal processes formation with distinct actin structures. However, it is largely unknown how cytoskeleton regulators differentially regulate microtubule organization in axon and dendrite development. Here, we characterize the role of actin regulators in axon and dendrite development and show that the RacGEF TIAM-1 regulates dendritic patterns through its N-terminal domains and suppresses axon growth through its C-terminal domains. TIAM-1 maintains plus-end-out microtubule orientation in posterior dendrites and prevents the accumulation of microtubules in the axon. In somatodendritic regions, TIAM-1 interacts with UNC-119 and stabilizes the organization between actin filaments and microtubules. UNC-119 is required for TIAM-1 to control axon growth, and its expression levels determine axon length. Taken together, TIAM-1 regulates neuronal microtubule organization and patterns axon and dendrite development respectively through its different domains.
Assuntos
Actinas , Dendritos , Dendritos/genética , Dendritos/metabolismo , Actinas/metabolismo , Axônios/metabolismo , Microtúbulos/metabolismo , Neurogênese/genéticaRESUMO
To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations.
Assuntos
Ciclo Celular/genética , Diferenciação Celular/genética , Proteínas de Drosophila/genética , Proteínas do Grupo Polycomb/genética , Fatores de Transcrição/genética , Animais , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Chaperonas de Histonas/biossíntese , Chaperonas de Histonas/genética , Histonas/genética , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Mutação , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas do Grupo Polycomb/biossíntese , Fatores de Transcrição/biossínteseRESUMO
Ribosome biogenesis takes place in the nucleolus, the size of which is often coordinated with cell growth and development. However, how metazoans control nucleolar size remains largely unknown. Caenorhabditis elegans provides a good model to address this question owing to distinct tissue distribution of nucleolar sizes and a mutant, ncl-1, which exhibits larger nucleoli than wild-type worms. Here, through a series of loss-of-function analyses, we report that the nucleolar size is regulated by a circuitry composed of microRNA let-7, translation repressor NCL-1, and a major nucleolar pre-rRNA processing protein FIB-1/fibrillarin. In cooperation with RNA binding proteins PUF and NOS, NCL-1 suppressed the translation of FIB-1/fibrillarin, while let-7 targeted the 3'UTR of ncl-1 and inhibited its expression. Consequently, the abundance of FIB-1 is tightly controlled and correlated with the nucleolar size. Together, our findings highlight a novel genetic cascade by which post-transcriptional regulators interplay in developmental control of nucleolar size and function.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Proteínas Cromossômicas não Histona/genética , MicroRNAs/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Regiões 3' não Traduzidas , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Nucléolo Celular/genética , Tamanho Celular , Proteínas Cromossômicas não Histona/metabolismo , Feminino , MicroRNAs/metabolismo , Imagem Óptica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Vulva/crescimento & desenvolvimento , Vulva/metabolismoRESUMO
Primary microRNAs (pri-miRNAs) are cleaved by the nuclear RNase III Drosha to produce hairpin-shaped precursor miRNAs (pre-miRNAs). In humans, this process is known to be facilitated by the DEAD-box helicases p68 (DDX5) and p72 (DDX17). In this study, we performed a candidate-based RNAi screen in C. elegans to identify DEAD/H-box proteins involved in miRNA biogenesis. In a let-7(mg279) sensitized genetic background, knockdown of a homolog of yeast splicing factor Prp28p, DDX-23, or a homolog of human helicases p68 and p72, DDX-17, enhanced let-7 loss-of-function phenotypes, suggesting that these helicases play a role in let-7 processing and/or function. In both ddx-23(RNAi) and ddx-17(RNAi), levels of mature let-7 were decreased while pri-let-7 was found to accumulate, indicating that the helicases likely act at the level of pri-let-7 processing. DDX-23 and DDX-17 were also required for the biogenesis of other known heterochronic miRNAs, including lin-4 and the let-7 family members miR-48, miR-84 and miR-241. Their function was not confined to the heterochronic pathway, however, since they were both necessary for down-regulation of cog-1 by the spatial patterning miRNA, lsy-6. Here, we present a novel function for C. elegans DDX-23 in pri-miRNA processing, and also suggest a conserved role for DDX-17 in this process.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , RNA Helicases DEAD-box/metabolismo , MicroRNAs/genética , Processamento Pós-Transcricional do RNA/genética , Animais , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Larva/genética , MicroRNAs/metabolismo , Mutação/genética , Interferência de RNARESUMO
The RNA-binding protein LIN28B represses the biogenesis of the tumor suppressor let-7. The LIN28B/let-7 axis regulates cell differentiation and is associated with various cancers. The RNA-binding protein Q (hnRNP Q) or SYNCRIP (Synaptotagmin Binding Cytoplasmic RNA Interacting Protein) has been implicated in mRNA splicing, mRNA transport, translation, and miRNAs biogenesis as well as metabolism in cancer. To determine whether hnRNP Q plays a role in the LIN28B/let-7 axis, we tested for interactions between hnRNP Q and LIN28B. We demonstrated that hnRNP Q interacts with LIN28B in an RNA-dependent manner. Knockdown of hnRNP Q caused reduced expression of a well-known let-7 target TRIM71, an E3 ubiquitin ligase that belongs to the RBCC/TRIM family, and also LIN28B, whose mRNA itself is down-regulated by let-7. In addition, hnRNP Q knockdown increased let-7 family miRNA levels and reduced the activity of luciferase reporters fused with the TRIM71 3'UTR or a synthetic 3'UTR carrying 8X let-7 complementary sites. Finally, depletion of hnRNP Q inhibited the proliferation of a hepatocellular carcinoma cell line, Huh7. This observation is consistent with the survival curve for liver cancer patients from the TCGA database, which indicates that high expression of hnRNP Q is a prognostic marker for a poor outcome in individuals afflicted with hepatocellular carcinoma. Together, our findings suggest that hnRNP Q interacts with LIN28B and modulates the LIN28B/let-7 axis in hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular , Ribonucleoproteínas Nucleares Heterogêneas , Neoplasias Hepáticas , MicroRNAs , Proteínas de Ligação a RNA , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Ligação Proteica , Regiões 3' não TraduzidasRESUMO
Mirtrons are short hairpin introns recently found in flies and nematodes that provide an alternative source for animal microRNA biogenesis and use the splicing machinery to bypass Drosha cleavage in initial maturation. The presence of mirtrons outside of invertebrates was not previously known. In the October 26 issue of Molecular Cell, Berezikov et al. expose a number of short mammalian introns as mirtrons.
Assuntos
Íntrons , MicroRNAs/fisiologia , Processamento Pós-Transcricional do RNA , Splicing de RNA , Animais , Humanos , MicroRNAs/genéticaRESUMO
Caenorhabditis elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination-mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to â¼33.4 kb. Taking advantage of this integration method we establish a dual-color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.
Assuntos
Bacteriófagos , Proteínas de Caenorhabditis elegans , Animais , Bacteriófagos/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Integrases/genética , Proteínas do Tecido Nervoso/genética , Recombinação Genética , TransgenesRESUMO
The areas where dengue virus (DENV) is endemic have expanded rapidly, driven in part by the global spread of Aedes species, which act as disease vectors. DENV replicates in the mosquito midgut and is disseminated to the mosquito's salivary glands for amplification. Thus, blocking virus infection or replication in the tissues of the mosquito may be a viable strategy for reducing the incidence of DENV transmission to humans. Here we used the mariner Mos1 transposase to create an Aedes aegypti line that expresses virus-specific miRNA hairpins capable of blocking DENV replication. These microRNA are driven by the blood-meal-inducible carboxypeptidase A promoter or by the polyubiquitin promoter. The transgenic mosquitoes exhibited significantly lower infection rates and viral titers for most DENV serotypes 7 days after receiving an infectious blood meal. The treatment was also effective at day 14 post infection after a second blood meal had been administered. In viral transmission assay, we found there was significantly reduced transmission in these lines. These transgenic mosquitoes were effective in silencing most of the DENV genome; such an approach may be employed to control a dengue fever epidemic.
Assuntos
Aedes/virologia , Animais Geneticamente Modificados , Vírus da Dengue/patogenicidade , Dengue/prevenção & controle , Controle de Mosquitos/métodos , Mosquitos Vetores/virologia , Aedes/genética , Animais , Linhagem Celular , Cricetinae , Cricetulus , Dengue/transmissão , Vírus da Dengue/genética , Fibroblastos/virologia , Mosquitos Vetores/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sorogrupo , Transposases/genética , Transposases/metabolismo , Carga ViralRESUMO
Increased levels of dysfunctional mitochondria within skeletal muscle are correlated with numerous age-related physiopathological conditions. Improving our understanding of the links between mitochondrial function and muscle proteostasis, and the role played by individual genes and regulatory networks, is essential to develop treatments for these conditions. One potential player is the mitochondrial outer membrane protein Fis1, a crucial fission factor heavily involved in mitochondrial dynamics in yeast but with an unknown role in higher-order organisms. By using Drosophila melanogaster as a model, we explored the effect of Fis1 mutations generated by transposon Minos-mediated integration. Mutants exhibited a higher ratio of damaged mitochondria with age as well as elevated reactive oxygen species levels compared with controls. This caused an increase in oxidative stress, resulting in large accumulations of ubiquitinated proteins, accelerated muscle function decline, and mitochondrial myopathies in young mutant flies. Ectopic expression of Fis1 isoforms was sufficient to suppress this phenotype. Loss of Fis1 led to unbalanced mitochondrial proteostasis within fly muscle, decreasing both flight capabilities and lifespan. Fis1 thus clearly plays a role in fly mitochondrial dynamics. Further investigations into the detailed function of Fis1 are necessary for exploring how mitochondrial function correlates with muscle health during aging.
Assuntos
Drosophila melanogaster/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Proteostase/genética , Envelhecimento , AnimaisRESUMO
The let-7 microRNA (miRNA) regulates developmental timing at the larval-to-adult transition in Caenorhabditis elegans. Dysregulation of let-7 results in irregular hypodermal and vulval development. Disrupted let-7 function is also a feature of human lung cancer. However, little is known about the mechanism and co-factors of let-7. Here we demonstrate that ribosomal protein RPS-14 is able to modulate let-7 function in C. elegans. The RPS-14 protein co-immunoprecipitated with the nematode Argonaute homolog, ALG-1. Reduction of rps-14 gene expression by RNAi suppressed the aberrant vulva and hypodermis development phenotypes of let-7(n2853) mutant animals and the mis-regulation of a reporter bearing the lin-41 3'UTR, a well established let-7 target. Our results indicate an interactive relationship between let-7 miRNA function and ribosomal protein RPS-14 in regulation of terminal differentiation that may help in understanding the mechanism of translational control by miRNAs.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , MicroRNAs/fisiologia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Caenorhabditis elegans/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Helmintos , MicroRNAs/genética , Mutação , FenótipoRESUMO
Little is known about the protein complexes required for microRNA formation and function. Here we used native gel electrophoresis to identify miRNA ribonucleoprotein complexes (miRNPs) in Caenorhabditis elegans. Our data reveal multiple distinct miRNPs that assemble on the let-7 miRNA in vitro. The formation of these complexes is affected but not abolished by alg-1 or alg-2 null mutations. The largest complex (M*) with an estimated molecular mass of >669 kDa cofractionates with the known RISC factors ALG-1, VIG-1, and TSN-1. The M* complex and two complexes, M3 and M4, with similar molecular weights of approximately 500 kDa, also assemble on all other miRNAs used in our experiments. Two smaller complexes, M1 (approximately 160 kDa) and M2 (approximately 250 kDa), assemble on the members of the let-7 miRNAs family but not lin-4 or mir-234, and their formation is highly dependent on specific sequences in the 5' seed region of let-7. Moreover, an unidentified protein, p40, which only appears in the M1 and M2 complexes, was detected by UV triggered cross-linking to let-7 but not to lin-4. The cross-linking of p40 to let-7 is also dependent on the let-7 sequence. Another unidentified protein, p13, is detected in all let-7 binding complexes and lin-4 cross-linked products. Our data suggest that besides being present in certain large miRNPs with sizes similar to reported RISC, the let-7 miRNA also assembles with specific binding proteins and forms distinct small complexes.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , MicroRNAs/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Mutação , Complexo de Inativação Induzido por RNA/genética , Ribonucleoproteínas/genéticaRESUMO
Cytoplasmic processing bodies, or P-bodies, contain a high concentration of enzymes and factors required for mRNA turnover and translational repression. Recent studies provide evidence that the mRNAs silenced by miRNAs are localized to P-bodies for storage or degradation, perhaps in adjacent subcompartments. mRNP remodeling, potentially induced by miRISC or RNA helicase activity, may cause the modification of the translation initiation complex at the 5' end of mRNA, following translational repression and localization to P-bodies. Further remodeling in P-bodies may facilitate access of the decapping complex to the cap structure, thus inducing mRNA degradation. However, with appropriate signals, stored mRNAs in P-bodies could be released and returned to the translational machinery through mechanisms requiring binding of regulatory proteins to the 3' UTR of mRNAs. Here a model is proposed to explain the repression and degradation stages of the mRNAs within PBs. This model includes preservation or disruption of a stable closed loop structure of the mRNAs, compartmentalization in PBs and mRNA escape triggered by additional binding proteins.
Assuntos
Estruturas Citoplasmáticas/genética , Inativação Gênica , MicroRNAs/fisiologia , Modelos Biológicos , Compartimento Celular , RNA Helicases , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNARESUMO
Exploiting a C. elegans mutant (ncl-1) exhibiting nucleolar abnormalities, we recently identified the let-7/ncl-1/fib-1 genetic cascade underlying proper rRNA abundance and nucleolar size. These 3 factors, let-7 (a miRNA), NCL-1 (a member of the TRIM-NHL family), and fibrillarin (a nucleolar methyltransferase), are evolutionarily conserved across metazoans. In this article, we provide several lines of bioinformatic evidence showing that human and Drosophila homologues of C. elegans NCL-1, TRIM-71 and Brat, respectively, likely act as translational suppressors of fibrillarin. Moreover, since their 3'-UTRs contain putative target sites, they may also be under the control of the let-7 miRNA. We hypothesize that let-7, TRIM and fibrillarin contribute activities in concert, and constitute a conserved network controlling nucleolar size in eukaryotes. We provide an in-depth literature review of various molecular pathways, including the let-7/ncl-1/fib-1 genetic cascade, implicated in the regulation of nucleolar size.
Assuntos
Nucléolo Celular , Evolução Molecular , Tamanho das Organelas/genética , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , HumanosRESUMO
Previously, we showed that BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing. In this study, we provide strong evidence that BCAS2 regulates the activity of Delta-Notch signaling via Delta pre-mRNA splicing. Depletion of dBCAS2 reduces Delta mRNA expression and leads to accumulation of Delta pre-mRNA, resulting in diminished transcriptions of Delta-Notch signaling target genes, such as cut and E(spl)m8. Furthermore, ectopic expression of human BCAS2 (hBCAS2) and Drosophila BCAS2 (dBCAS2) in a dBCAS2-deprived fly can rescue dBCAS2 depletion-induced wing damage to the normal phenotypes. These rescued phenotypes are correlated with the restoration of Delta pre-mRNA splicing, which affects Delta-Notch signaling activity. Additionally, overexpression of Delta can rescue the wing deformation by deprivation of dBCAS2; and the depletion of dBCAS2 can restore the aberrant eye associated with Delta-overexpressing retinas; providing supporting evidence for the regulation of Delta-Notch signaling by dBCAS2. Taken together, dBCAS2 participates in Delta pre-mRNA splicing that affects the regulation of Delta-Notch signaling in Drosophila wing development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Neoplasias/metabolismo , Precursores de RNA/metabolismo , Receptores Notch/metabolismo , Animais , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Olho/metabolismo , Humanos , Proteínas de Neoplasias/genética , Fenótipo , Plasmídeos/genética , Plasmídeos/metabolismo , Precursores de RNA/genética , Splicing de RNA , Receptores Notch/genética , Transdução de Sinais , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
The let-7 microRNA (miRNA) regulates cell cycle exit and terminal differentiation in the C. elegans heterochronic gene pathway. Low expression of let-7 results in retarded vulva and hypodermal cell development in C. elegans and has been associated with several human cancers. Previously, the versatile scaffold protein receptor for activated C kinase 1 (RACK1) was proposed to facilitate recruitment of the miRNA-induced silencing complex (miRISC) to the polysome and to be required for miRNA function in C. elegans and humans. Here, we show that depletion of C. elegans RACK-1 by RNAi increases let-7 miRNA levels and suppresses the retarded terminal differentiation of lateral hypodermal seam cells in mutants carrying the hypomorphic let-7(n2853) allele or lacking the let-7 family miRNA genes mir-48 and mir-241. Depletion of RACK-1 also increases the levels of precursor let-7 miRNA. When Dicer is knocked down and pre-miRNA processing is inhibited, depletion of RACK-1 still leads to increased levels of pre-let-7, suggesting that RACK-1 affects a biogenesis mechanism upstream of Dicer. No changes in the activity of the let-7 promoter or the levels of primary let-7 miRNA are associated with depletion of RACK-1, suggesting that RACK-1 affects let-7 miRNA biogenesis at the post-transcriptional level. Interestingly, rack-1 knockdown also increases the levels of a few other precursor miRNAs. Our results reveal that RACK-1 controls the biogenesis of a subset of miRNAs, including let-7, and in this way plays a role in the heterochronic gene pathway during C. elegans development.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Diferenciação Celular , MicroRNAs/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , MicroRNAs/metabolismo , Mutação , Interferência de RNA , Receptores Citoplasmáticos e Nucleares/genética , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
Originally discovered in C. elegans, microRNAs (miRNAs) are small RNAs that regulate fundamental cellular processes in diverse organisms. MiRNAs are encoded within the genome and are initially transcribed as primary transcripts that can be several kilobases in length. Primary transcripts are successively cleaved by two RNase III enzymes, Drosha in the nucleus and Dicer in the cytoplasm, to produce â¼70 nucleotide (nt) long precursor miRNAs and 22 nt long mature miRNAs, respectively. Mature miRNAs regulate gene expression post-transcriptionally by imperfectly binding target mRNAs in association with the multiprotein RNA induced silencing complex (RISC). The conserved sequence, expression pattern, and function of some miRNAs across distinct species as well as the importance of specific miRNAs in many biological pathways have led to an explosion in the study of miRNA biogenesis, miRNA target identification, and miRNA target regulation. Many advances in our understanding of miRNA biology have come from studies in the powerful model organism C. elegans. This chapter reviews the current methods used in C. elegans to study miRNA biogenesis, small RNA populations, miRNA-protein complexes, and miRNA target regulation.
Assuntos
Caenorhabditis elegans/genética , Expressão Gênica , MicroRNAs/genética , RNA de Helmintos/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica , Genes Reporter , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , RNA de Helmintos/isolamento & purificação , RNA de Helmintos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNARESUMO
The Prp19-associated complex, consisting of at least eight protein components, is involved in spliceosome activation by specifying the interaction of U5 and U6 with pre-mRNA for their stable association with the spliceosome after U4 dissociation. We show here that yeast cells depleted of one or two of the Prp19-associated components, accumulate the free form of U4. In NTC25-deleted cells, the level of U6 was also reduced. Extracts prepared from NTC25-deleted cells contained neither free U4 nor U6 and were ineffective in spliceosome recycling in the in vitro splicing reaction. Overexpression of U6 partially rescued the temperature-sensitive growth defect and decreased the relative amount of free U4 in NTC25-deleted cells, indicating that the accumulation of free U4 was a consequence of insufficient amounts of U6 snRNA. Extracts prepared from U6-overproducing NTC25-deleted cells containing free-form U6 were capable of spliceosome recycling, suggesting a role of free U6 RNP in spliceosome recycling. Our results demonstrate that in addition to direct participation in spliceosome activation, the Prp19-associated complex has an indirect role in spliceosome recycling through affecting the biogenesis of U4/U6 snRNP in the in vivo splicing reaction.
Assuntos
Ribonucleoproteína Nuclear Pequena U4-U6 , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos , Northern Blotting , Western Blotting , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Genes Fúngicos , Modelos Biológicos , Plasmídeos/genética , Splicing de RNA , Fatores de Processamento de RNA , RNA Mensageiro/análise , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Spliceossomos/química , Spliceossomos/metabolismoRESUMO
Activation of the spliceosome involves a major structural change in the spliceosome, including release of U1 and U4 small nuclear ribonucleoprotein particles and the addition of a large protein complex, the Prp19-associated complex. We previously showed that the Prp19-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is released. Changes within the spliceosome upon binding of the Prp19-associated complex include remodeling of the U6/5' splice site interaction and destabilization of Lsm proteins to allow further interaction of U6 with the intron sequence. Here, we further analyzed interactions of U5 and U6 with pre-mRNA at various stages of spliceosome assembly from initial binding of tri-small nuclear ribonucleoprotein complex to the activated spliceosome to reveal stepwise changes of interactions. We demonstrate that both U5 and U6 interacted with pre-mRNA in dynamic manners spanning over a large region of U6 and the 5' exon sequences prior to the activation of the spliceosome. During spliceosome activation, interactions were locked down to small regions, and the Prp19-associated complex was required for defining the specificity of interaction of U5 and U6 with the 5' splice site to stabilize their association with the spliceosome after U4 is dissociated.
Assuntos
Precursores de RNA/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Spliceossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Pareamento de Bases , Sequência de Bases , Dados de Sequência Molecular , Complexos Multiproteicos , Conformação de Ácido Nucleico , Ligação Proteica , Fatores de Processamento de RNA , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
During spliceosome activation, a large structural rearrangement occurs that involves the release of two small nuclear RNAs, U1 and U4, and the addition of a protein complex associated with Prp19p. We show here that the Prp19p-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is dissociated. Ultraviolet crosslinking analysis revealed the existence of two modes of base pairing between U6 and the 5' splice site, as well as a switch of such base pairing from one to the other that required the Prp19p-associated complex during spliceosome activation. Moreover, a Prp19p-dependent structural change in U6 small nuclear ribonucleoprotein particles was detected that involves destabilization of Sm-like (Lsm) proteins to bring about interactions between the Lsm binding site of U6 and the intron sequence near the 5' splice site, indicating dynamic association of Lsm with U6 and a direct role of Lsm proteins in activation of the spliceosome.