RESUMO
Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here, we overcome these hurdles through the development of "Ribo-FilterOut", which is based on the separation of footprints from ribosome subunits by ultrafiltration, and "Ribo-Calibration", which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method reveals the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro , Ribossomos , Ribossomos/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Humanos , Transcriptoma , RNA Ribossômico/metabolismo , RNA Ribossômico/genética , Sequenciamento de Nucleotídeos em Larga Escala , Calibragem , Resposta ao Choque Térmico/genética , Perfil de RibossomosRESUMO
Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, we here revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5' leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open-reading frames in the 5' leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.
Assuntos
Neuroglia , Biossíntese de Proteínas , Animais , Neuroglia/metabolismo , Neurônios/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Regulação da Expressão Gênica , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Encéfalo/metabolismo , Encéfalo/citologia , Ribossomos/metabolismo , Drosophila/genéticaRESUMO
Neat1 is an architectural RNA that provides the structural basis for nuclear bodies known as paraspeckles. Although the assembly processes by which Neat1 organizes paraspeckle components are well-documented, the physiological functions of Neat1 are not yet fully understood. This is partly because Neat1 knockout (KO) mice, lacking paraspeckles, do not exhibit overt phenotypes under normal laboratory conditions. During our search for conditions that elicit clear phenotypes in Neat1 KO mice, we discovered that the differentiation of beige adipocytes-inducible thermogenic cells that emerge upon cold exposure-is severely impaired in these mutant mice. Neat1_2, the architectural isoform of Neat1, is transiently upregulated during the early stages of beige adipocyte differentiation, coinciding with increased paraspeckle formation. Genes with altered expression during beige adipocyte differentiation typically cluster at specific chromosomal locations, some of which move closer to paraspeckles upon cold exposure. These observations suggest that paraspeckles might coordinate the regulation of these gene clusters by controlling the activity of certain transcriptional condensates that coregulate multiple genes. We propose that our findings highlight a potential role for Neat1 and paraspeckles in modulating chromosomal organization and gene expression, potentially crucial processes for the differentiation of beige adipocytes.
Assuntos
Adipócitos Bege , Diferenciação Celular , Temperatura Baixa , Camundongos Knockout , RNA Longo não Codificante , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Camundongos , Diferenciação Celular/genética , Adipócitos Bege/metabolismo , Adipócitos Bege/citologia , Termogênese/genéticaRESUMO
Eukaryotic translation initiation factor (eIF)4A-a DEAD-box RNA-binding protein-plays an essential role in translation initiation. Recent reports have suggested helicase-dependent and helicase-independent functions for eIF4A, but the multifaceted roles of eIF4A have not been fully explored. Here we show that eIF4A1 enhances translational repression during the inhibition of mechanistic target of rapamycin complex 1 (mTORC1), an essential kinase complex controlling cell proliferation. RNA pulldown followed by sequencing revealed that eIF4A1 preferentially binds to mRNAs containing terminal oligopyrimidine (TOP) motifs, whose translation is rapidly repressed upon mTORC1 inhibition. This selective interaction depends on a La-related RNA-binding protein, LARP1. Ribosome profiling revealed that deletion of EIF4A1 attenuated the translational repression of TOP mRNAs upon mTORC1 inactivation. Moreover, eIF4A1 increases the interaction between TOP mRNAs and LARP1 and, thus, ensures stronger translational repression upon mTORC1 inhibition. Our data show the multimodality of eIF4A1 in modulating protein synthesis through an inhibitory binding partner and provide a unique example of the repressive role of a universal translational activator.
RESUMO
Gene expression is known to vary among individuals, and this variability can impact the phenotypic diversity observed in natural populations. While the transcriptome and proteome have been extensively studied, little is known about the translation process itself. Here, we therefore performed ribosome and transcriptomic profiling on a genetically and ecologically diverse set of natural isolates of the Saccharomyces cerevisiae yeast. Interestingly, we found that the Euclidean distances between each profile and the expression fold changes in each pairwise isolate comparison were higher at the transcriptomic level. This observation clearly indicates that the transcriptional variation observed in the different isolates is buffered through a phenomenon known as post-transcriptional buffering at the translation level. Furthermore, this phenomenon seemed to have a specific signature by preferentially affecting essential genes as well as genes involved in complex-forming proteins, and low transcribed genes. We also explored the translation of the S. cerevisiae pangenome and found that the accessory genes related to introgression events displayed similar transcription and translation levels as the core genome. By contrast, genes acquired through horizontal gene transfer events tended to be less efficiently translated. Together, our results highlight both the extent and signature of the post-transcriptional buffering.
Assuntos
Saccharomyces cerevisiae , Transcriptoma , Humanos , Saccharomyces cerevisiae/genética , Perfilação da Expressão Gênica , Ribossomos/genética , Patrimônio Genético , Variação GenéticaRESUMO
4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.
Assuntos
Splicing de RNA , Pequeno RNA não Traduzido , Camundongos , Animais , Splicing de RNA/genética , Éxons/genética , Retroelementos/genética , Códon sem Sentido , Processamento AlternativoRESUMO
Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.
Assuntos
RNA de Transferência , Animais , Humanos , Ratos , Anticódon , Linhagem Celular , Códon , Glicosilação , Nucleosídeo Q/química , Nucleosídeo Q/genética , Nucleosídeo Q/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Suínos , Peixe-Zebra/metabolismo , Conformação de Ácido NucleicoRESUMO
The prion-like domain (PrLD) is a class of intrinsically disordered regions. Although its propensity to form condensates has been studied in the context of neurodegenerative diseases, the physiological role of PrLD remains unclear. Here, we investigated the role of PrLD in the RNA-binding protein NFAR2, generated by a splicing variant of the Ilf3 gene. Removal of the PrLD in mice did not impair the function of NFAR2 required for survival, but did affect the responses to chronic water immersion and restraint stress (WIRS). The PrLD was required for WIRS-sensitive nuclear localization of NFAR2 and WIRS-induced changes in mRNA expression and translation in the amygdala, a fear-related brain region. Consistently, the PrLD conferred resistance to WIRS in fear-associated memory formation. Our study provides insights into the PrLD-dependent role of NFAR2 for chronic stress adaptation in the brain.
RESUMO
Plants often generate secondary metabolites as defense mechanisms against parasites. Although some fungi may potentially overcome the barrier presented by antimicrobial compounds, only a limited number of examples and molecular mechanisms of resistance have been reported. Here, we found an Aglaia plant-parasitizing fungus that overcomes the toxicity of rocaglates, which are translation inhibitors synthesized by the plant, through an amino acid substitution in a eukaryotic translation initiation factor (eIF). De novo transcriptome assembly revealed that the fungus belongs to the Ophiocordyceps genus and that its eIF4A, a molecular target of rocaglates, harbors an amino acid substitution critical for rocaglate binding. Ribosome profiling harnessing a cucumber-infecting fungus, Colletotrichum orbiculare, demonstrated that the translational inhibitory effects of rocaglates were largely attenuated by the mutation found in the Aglaia parasite. The engineered C. orbiculare showed a survival advantage on cucumber plants with rocaglates. Our study exemplifies a plant-fungus tug-of-war centered on secondary metabolites produced by host plants.
Although plants may seem like passive creatures, they are in fact engaged in a constant battle against the parasitic fungi that attack them. To combat these fungal foes, plants produce small molecules that act like chemical weapons and kill the parasite. However, the fungi sometimes fight back, often by developing enzymes that can break down the deadly chemicals into harmless products. One class of anti-fungal molecules that has drawn great interest is rocaglates, as they show promise as treatments for cancer and COVID-19. Rocaglates are produced by plants in the Aglaia family and work by targeting the fungal molecule eIF4A which is fundamental for synthesizing proteins. Since proteins perform most of the chemistry necessary for life, one might think that rocaglates could ward off any fungus. But Chen et al. discovered there is in fact a species of fungi that can evade this powerful defense mechanism. After seeing this new-found fungal species successfully growing on Aglaia plants, Chen et al. set out to find how it is able to protect itself from rocoglates. Genetic analysis of the fungus revealed that its eIF4A contained a single mutation that 'blocked' rocaglates from interacting with it. Chen et al. confirmed this effect by engineering a second fungal species (which infects cucumber plants) so that its elF4A protein contained the mutation found in the new fungus. Fungi with the mutated eIF4A thrived on cucumber leaves treated with a chemical derived from rocaglates, whereas fungi with the non-mutated version were less successful. These results shed new light on the constant 'arms race' between plants and their fungal parasites, with each side evolving more sophisticated ways to overcome the other's defenses. Chen et al. hope that identifying the new rocaglate-resistant eIF4A mutation will help guide the development and use of any therapies based on rocaglates. Further work investigating how often the mutation occurs in humans will also be important for determining how effective these therapies will be.
Assuntos
Aglaia , Hypocreales , Parasitos , Animais , Substituição de Aminoácidos , MutaçãoRESUMO
Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2 The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2 that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.
Assuntos
Paraspeckles , RNA Longo não Codificante , Animais , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Ratos-Toupeira/genética , Ratos-Toupeira/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Chemical splicing modulators that bind to the spliceosome have provided an attractive avenue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. However, the biological effect of proteins containing translated intron sequences remains unclear. Here, we identify a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) via genome-wide ribosome profiling and bio-orthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through c-Jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Splicing de RNA/genética , Spliceossomos/genética , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Íntrons , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Piranos/farmacologia , Splicing de RNA/efeitos dos fármacos , RNA-Seq , Compostos de Espiro/farmacologia , Spliceossomos/efeitos dos fármacosRESUMO
Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2Bâ¢SFSV NSsâ¢unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.
Assuntos
Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Doenças dos Animais , Animais , Linhagem Celular , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/genética , Feminino , Humanos , Modelos Moleculares , Neurônios , Phlebovirus , Fosforilação , Ligação Proteica , Ratos , Ratos Wistar , Ribossomos , Proteínas Virais/genéticaRESUMO
The translation inhibitor rocaglamide A (RocA) has shown promising antitumor activity because it uniquely clamps eukaryotic initiation factor (eIF) 4A onto polypurine RNA for selective translational repression. As eIF4A has been speculated to be a unique target of RocA, alternative targets have not been investigated. Here, we reveal that DDX3 is another molecular target of RocA. Proximity-specific fluorescence labeling of an O-nitrobenzoxadiazole-conjugated derivative revealed that RocA binds to DDX3. RocA clamps the DDX3 protein onto polypurine RNA in an ATP-independent manner. Analysis of a de novo-assembled transcriptome from the plant Aglaia, a natural source of RocA, uncovered the amino acid critical for RocA binding. Moreover, ribosome profiling showed that because of the dominant-negative effect of RocA, high expression of eIF4A and DDX3 strengthens translational repression in cancer cells. This study indicates that sequence-selective clamping of DDX3 and eIF4A, and subsequent dominant-negative translational repression by RocA determine its tumor toxicity.
Assuntos
Benzofuranos/farmacologia , RNA Helicases DEAD-box/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Benzofuranos/química , Células Cultivadas , RNA Helicases DEAD-box/metabolismo , Inibidores Enzimáticos/química , Fator de Iniciação 4A em Eucariotos/metabolismo , Feminino , Humanos , Masculino , Modelos Moleculares , Conformação MolecularRESUMO
Ribosomes often encounter obstacles during translation elongation and thus collide with each other. Disome profiling, an optimized ribosome profiling method, specifically sequences the long ribosome footprints generated from collided ribosomes produced by the ribosome pause and thus allows the survey of sites in a genome-wide manner. This protocol details the procedure from lysate preparation of human tissue cultures and zebrafish embryos to sequencing library construction. For complete details on the use and execution of this protocol, please refer to Han et al. (2020).
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ribossomos/metabolismo , Peixe-Zebra/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Humanos , Reação em Cadeia da Polimerase , RNA Guia de Cinetoplastídeos/metabolismo , RNA Ribossômico/metabolismoRESUMO
Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.
Assuntos
Processamento Pós-Transcricional do RNA , RNA Mitocondrial/química , RNA de Transferência/química , Feminino , Código Genético , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Estrutura Molecular , Nucleosídeo Q/biossíntese , Nucleosídeo Q/química , Fosforilação Oxidativa , Placenta , Gravidez , RNA Mitocondrial/isolamento & purificação , RNA Mitocondrial/metabolismo , RNA de Transferência/isolamento & purificação , RNA de Transferência/metabolismo , RNA-SeqRESUMO
Ribosome movement is not always smooth and is rather often impeded. For ribosome pauses, fundamental issues remain to be addressed, including where ribosomes pause on mRNAs, what kind of RNA/amino acid sequence causes this pause, and the physiological significance of this attenuation of protein synthesis. Here, we survey the positions of ribosome collisions caused by ribosome pauses in humans and zebrafish using modified ribosome profiling. Collided ribosomes, i.e., disomes, emerge at various sites: Pro-Pro/Gly/Asp motifs; Arg-X-Lys motifs; stop codons; and 3' untranslated regions. The electrostatic interaction between the charged nascent chain and the ribosome exit tunnel determines the eIF5A-mediated disome rescue at the Pro-Pro sites. In particular, XBP1u, a precursor of endoplasmic reticulum (ER)-stress-responsive transcription factor, shows striking queues of collided ribosomes and thus acts as a degradation substrate by ribosome-associated quality control. Our results provide insight into the causes and consequences of ribosome pause by dissecting collided ribosomes.
Assuntos
Códon de Terminação/genética , Biossíntese de Proteínas/genética , Ribossomos/genética , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Códon de Terminação/metabolismo , Humanos , Elongação Traducional da Cadeia Peptídica/genética , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Peixe-ZebraRESUMO
Oxytocin is a central neuromodulator required for facilitating mate preferences for familiar individuals in a monogamous rodent (prairie vole), irrespective of sex. While the role of oxytocin in mate choice is only understood in a few monogamous species, its function in nonmonogamous species, comprising the vast majority of vertebrate species, remains unclear. To address this issue, we evaluated the involvement of an oxytocin homolog (isotocin, referred herein as oxt) in mate choice in medaka fish (Oryzias latipes). Female medaka prefer to choose familiar mates, whereas male medaka court indiscriminately, irrespective of familiarity. We generated mutants of the oxt ligand (oxt) and receptor genes (oxtr1 and oxtr2) and revealed that the oxt-oxtr1 signaling pathway was essential for eliciting female mate preference for familiar males. This pathway was also required for unrestricted and indiscriminate mating strategy in males. That is, either oxt or oxtr1 mutation in males decreased the number of courtship displays toward novel females, but not toward familiar females. Further, males with these mutations exhibited enhanced mate-guarding behaviors toward familiar females, but not toward novel females. In addition, RNA-sequencing (seq) analysis revealed that the transcription of genes involved in gamma-amino butyric acid metabolism as well as those encoding ion-transport ATPase are up-regulated in both oxt and oxtr1 mutants only in female medaka, potentially explaining the sex difference of the mutant phenotype. Our findings provide genetic evidence that oxt-oxtr1 signaling plays a role in the mate choice for familiar individuals in a sex-specific manner in medaka fish.
Assuntos
Preferência de Acasalamento Animal/fisiologia , Oryzias/genética , Oryzias/fisiologia , Ocitocina/genética , Ocitocina/fisiologia , Reprodução/fisiologia , Animais , Corte , Feminino , Masculino , Mutação , Ocitocina/análogos & derivados , Fenótipo , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Reconhecimento Psicológico , Reprodução/genética , Caracteres Sexuais , Comportamento Sexual Animal/fisiologiaRESUMO
Neat1 is a long noncoding RNA (lncRNA) that serves as an architectural component of the nuclear bodies known as paraspeckles. Two isoforms of Neat1, the short isoform Neat1_1 and the long isoform Neat1_2, are generated from the same gene locus by alternative 3' processing. Neat1_1 is the most abundant and the best conserved isoform expressed in various cell types, whereas Neat1_2 is expressed in a small population of particular cell types, including the tip cells of the intestinal epithelium. To investigate the physiological significance of isoform switching, we created mutant mice that solely expressed Neat1_2 by deleting the upstream polyadenylation (poly-A) signal (PAS) required for the production of Neat1_1. We observed the loss of Neat1_1 and strong up-regulation of Neat1_2 in various tissues and cells and the subsequent hyperformation of paraspeckles, especially in cells that normally express Neat1_2. However, the mutant mice were born at the expected Mendelian ratios and did not exhibit obvious external and histological abnormalities. These observations suggested that the hyperformation of paraspeckles does not interfere with the development and growth of these animals under normal laboratory conditions.
Assuntos
Linhagem da Célula/genética , Isoformas de RNA/genética , RNA Longo não Codificante/genética , Células 3T3 , Animais , Regulação da Expressão Gênica/genética , Camundongos , Mutação/genética , Sinais de Poliadenilação na Ponta 3' do RNA/genéticaRESUMO
NEAT1 is one of the most studied lncRNAs, in part because its silencing in mice causes defects in mammary gland development and corpus luteum formation and protects them from skin cancer development. Moreover, depleting NEAT1 in established cancer cell lines reduces growth and sensitizes cells to DNA damaging agents. However, NEAT1 produces two isoforms and because the short isoform, NEAT1_1, completely overlaps the 5' part of the long NEAT1_2 isoform; the respective contributions of each of the isoforms to these phenotypes has remained unclear. Whereas NEAT1_1 is highly expressed in most tissues, NEAT1_2 is the central architectural component of paraspeckles, which are nuclear bodies that assemble in specific tissues and cells exposed to various forms of stress. Using dual RNA-FISH to detect both NEAT1_1 outside of the paraspeckles and NEAT1_2/NEAT1 inside this nuclear body, we report herein that NEAT1_1 levels are dynamically regulated during the cell cycle and targeted for degradation by the nuclear RNA exosome. Unexpectedly, however, cancer cells engineered to lack NEAT1_1, but not NEAT1_2, do not exhibit cell cycle defects. Moreover, Neat1_1-specific knockout mice do not exhibit the phenotypes observed in Neat1-deficient mice. We propose that NEAT1 functions are mainly, if not exclusively, attributable to NEAT1_2 and, by extension, to paraspeckles.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , RNA Longo não Codificante/genética , Processamento Alternativo , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Exossomos/metabolismo , Técnicas de Inativação de Genes , Homeostase , Humanos , Hibridização in Situ Fluorescente , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Estabilidade de RNA , Estresse Fisiológico/genética , TranscriptomaRESUMO
Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.