RESUMO
Visualizing the location and dynamics of RNAs in live cells is key to understanding their function. Here, we identify two endonuclease-deficient, single-component programmable RNA-guided and RNA-targeting Cas13 RNases (dCas13s) that allow robust real-time imaging and tracking of RNAs in live cells, even when using single 20- to 27-nt-long guide RNAs. Compared to the aptamer-based MS2-MCP strategy, an optimized dCas13 system is user friendly, does not require genetic manipulation, and achieves comparable RNA-labeling efficiency. We demonstrate that the dCas13 system is capable of labeling NEAT1, SatIII, MUC4, and GCN4 RNAs and allows the study of paraspeckle-associated NEAT1 dynamics. Applying orthogonal dCas13 proteins or combining dCas13 and MS2-MCP allows dual-color imaging of RNAs in single cells. Further combination of dCas13 and dCas9 systems allows simultaneous visualization of genomic DNA and RNA transcripts in living cells.
Assuntos
Imagem Molecular/métodos , RNA/fisiologia , Imagem Individual de Molécula/métodos , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Mucina-4 , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos/genética , RNA Longo não Codificante , Ribonucleases/genética , Ribonucleases/metabolismo , Coloração e Rotulagem/métodosRESUMO
Sequences of circular RNAs (circRNAs) produced from back-splicing of exon(s) completely overlap with those from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction (BSJ) sites. Therefore, examination of global circRNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites, which is different from the quantification of linear RNA expression by normalized RNA-seq fragments mapped to whole gene bodies. Thus, direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging. Here, we update the previously-reported CIRCexplorer pipeline to version 3 for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq (CIRCexplorer3-CLEAR). A new quantitation parameter, fragments per billion mapped bases (FPB), is applied to evaluate circular and linear RNA expression individually by fragments mapped to circRNA-specific BSJ sites or to linear RNA-specific splicing junction (SJ) sites. Comparison of circular and linear RNA expression levels is directly achieved by dividing FPBcirc by FPBlinear to generate a CIRCscore, which indicates the relative circRNA expression level using linear RNA expression level as the background. Highly-expressed circRNAs with low cognate linear RNA expression background can be readily identified by CIRCexplorer3-CLEAR for further investigation. CIRCexplorer3-CLEAR is publically available at https://github.com/YangLab/CLEAR.
Assuntos
RNA Circular/metabolismo , RNA/metabolismo , Interface Usuário-Computador , Linhagem Celular Tumoral , Humanos , RNA/química , Splicing de RNA , RNA Circular/química , Análise de Sequência de RNA , TranscriptomaRESUMO
Murine polyomavirus (MPyV) infects mouse cells and is highly oncogenic in immunocompromised hosts and in other rodents. Its genome is a small, circular DNA molecule of just over 5000 base pairs and it encodes only seven polypeptides. While seemingly simply organized, this virus has adopted an unusual genome structure and some unusual uses of cellular quality control pathways that, together, allow an amazingly complex and varied pattern of gene regulation. In this review we discuss how MPyV leverages these various pathways to control its life cycle.
Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Polyomavirus/imunologia , Polyomavirus/fisiologia , Replicação Viral , Animais , CamundongosRESUMO
DNA methylation is essential for mammalian development and physiology. Here we report that the developmentally regulated H19 lncRNA binds to and inhibits S-adenosylhomocysteine hydrolase (SAHH), the only mammalian enzyme capable of hydrolysing S-adenosylhomocysteine (SAH). SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases that methylate diverse cellular components, including DNA, RNA, proteins, lipids and neurotransmitters. We show that H19 knockdown activates SAHH, leading to increased DNMT3B-mediated methylation of an lncRNA-encoding gene Nctc1 within the Igf2-H19-Nctc1 locus. Genome-wide methylation profiling reveals methylation changes at numerous gene loci consistent with SAHH modulation by H19. Our results uncover an unanticipated regulatory circuit involving broad epigenetic alterations by a single abundantly expressed lncRNA that may underlie gene methylation dynamics of development and diseases and suggest that this mode of regulation may extend to other cellular components.
Assuntos
Adenosil-Homocisteinase/metabolismo , RNA Longo não Codificante/metabolismo , Adenosil-Homocisteinase/genética , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Genoma , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Ligação Proteica , RNA Longo não Codificante/genética , S-Adenosil-Homocisteína/metabolismo , DNA Metiltransferase 3BRESUMO
Mouse polyomavirus (MPyV) lytically infects mouse cells, transforms rat cells in culture, and is highly oncogenic in rodents. We have used deep sequencing to follow MPyV infection of mouse NIH3T6 cells at various times after infection and analyzed both the viral and cellular transcriptomes. Alignment of sequencing reads to the viral genome illustrated the transcriptional profile of the early-to-late switch with both early-strand and late-strand RNAs being transcribed at all time points. A number of novel insights into viral gene expression emerged from these studies, including the demonstration of widespread RNA editing of viral transcripts at late times in infection. By late times in infection, 359 host genes were seen to be significantly upregulated and 857 were downregulated. Gene ontology analysis indicated transcripts involved in translation, metabolism, RNA processing, DNA methylation, and protein turnover were upregulated while transcripts involved in extracellular adhesion, cytoskeleton, zinc finger binding, SH3 domain, and GTPase activation were downregulated. The levels of a number of long noncoding RNAs were also altered. The long noncoding RNA MALAT1, which is involved in splicing speckles and used as a marker in many late-stage cancers, was noticeably downregulated, while several other abundant noncoding RNAs were strongly upregulated. We discuss these results in light of what is currently known about the MPyV life cycle and its effects on host cell growth and metabolism.
Assuntos
Genoma Viral/genética , Interações Hospedeiro-Parasita/genética , Infecções por Polyomavirus/genética , Infecções Tumorais por Vírus/genética , Animais , Linhagem Celular , Camundongos , Polyomavirus/genética , Edição de RNA/genética , RNA Viral/genéticaRESUMO
The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant rodents. This decrease leads to increased bioavailability of let-7, causing diminished expression of let-7 targets, which is recapitulated in vitro where H19 depletion results in impaired insulin signaling and decreased glucose uptake. Furthermore, acute hyperinsulinemia downregulates H19, a phenomenon that occurs through PI3K/AKT-dependent phosphorylation of the miRNA processing factor KSRP, which promotes biogenesis of let-7 and its mediated H19 destabilization. Our results reveal a previously undescribed double-negative feedback loop between sponge lncRNA and target miRNA that contributes to glucose regulation in muscle cells.
Assuntos
Glucose/metabolismo , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Regulação para Baixo , Retroalimentação Fisiológica , Humanos , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Insulina/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas de Ligação a RNA/fisiologia , Transdução de Sinais , Transativadores/fisiologiaRESUMO
The introduction of double stranded RNA (dsRNA) into the cytoplasm of mammalian cells usually leads to a potent antiviral response resulting in the rapid induction of interferon beta (IFNß). This response can be mediated by a number of dsRNA sensors, including TLR3, MDA5, RIG-I and PKR. We show here that pluripotent human cells (human embryonic stem (hES) cells and induced pluripotent (iPS) cells) do not induce interferon in response to cytoplasmic dsRNA, and we have used a variety of approaches to learn the underlying basis for this phenomenon. Two major cytoplasmic dsRNA sensors, TLR3 and MDA5, are not expressed in hES cells and iPS cells. PKR is expressed in hES cells, but is not activated by transfected dsRNA. In addition, RIG-I is expressed, but fails to respond to dsRNA because its signaling adapter, MITA/STING, is not expressed. Finally, the interferon-inducible RNAse L and oligoadenylate synthetase enzymes are also expressed at very low levels. Upon differentiation of hES cells into trophoblasts, cells acquire the ability to respond to dsRNA and this correlates with a significant induction of expression of TLR3 and its adaptor protein TICAM-1/TRIF. Taken together, our results reveal that the lack of an interferon response may be a general characteristic of pluripotency and that this results from the systematic downregulation of a number of genes involved in cytoplasmic dsRNA signaling.
Assuntos
Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA de Cadeia Dupla/metabolismo , 2',5'-Oligoadenilato Sintetase/metabolismo , Citoplasma/metabolismo , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Helicase IFIH1 Induzida por Interferon , Interferon beta/genética , Interferon beta/metabolismo , Receptores Imunológicos , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Not all long, polyadenylated cellular RNAs encode polypeptides. In recent years, it has become apparent that a number of organisms express abundant amounts of transcripts that lack open reading frames or that are retained in the nucleus. Rather than accumulating silently in the cell, we now know that many of these long noncoding RNAs (lncRNAs) play important roles in nuclear architecture or in the regulation of gene expression. Here, we discuss some recent progress in our understanding of the functions of a number of important lncRNAs in mammalian cells.
Assuntos
Células/metabolismo , Mamíferos/genética , RNA não Traduzido/fisiologia , Animais , Sequência de Bases/fisiologia , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Humanos , Mamíferos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Especificidade de Órgãos/fisiologia , RNA não Traduzido/química , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Distribuição TecidualRESUMO
In many cells, mRNAs containing inverted repeats (Alu repeats in humans) in their 3' untranslated regions (3'UTRs) are inefficiently exported to the cytoplasm. Nuclear retention correlates with adenosine-to-inosine editing and is in paraspeckle-associated complexes containing the proteins p54(nrb), PSF, and PSP1 alpha. We report that robust editing activity in human embryonic stem cells (hESCs) does not lead to nuclear retention. p54(nrb), PSF, and PSP1 alpha are all expressed in hESCs, but paraspeckles are absent and only appear upon differentiation. Paraspeckle assembly and function depend on expression of a long nuclear-retained noncoding RNA, NEAT1. This RNA is not detectable in hESCs but is induced upon differentiation. Knockdown of NEAT1 in HeLa cells results both in loss of paraspeckles and in enhanced nucleocytoplasmic export of mRNAs containing inverted Alu repeats. Taken together, these results assign a biological function to a large noncoding nuclear RNA in the regulation of mRNA export.
Assuntos
Núcleo Celular/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Edição de RNA , RNA Mensageiro/metabolismo , RNA Nuclear/metabolismo , RNA não Traduzido/metabolismo , Regiões 3' não Traduzidas , Transporte Ativo do Núcleo Celular , Adenosina Desaminase/metabolismo , Elementos Alu , Sequência de Bases , Diferenciação Celular/genética , Estruturas do Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB , Interferência de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
Not only is gene regulation in polyoma interesting, but it has also proven to be highly informative and illustrative of a number of novel concepts in gene regulation. Of special interest and importance are the mechanisms by which this virus switches from the expression of early gene products to late gene products after the onset of viral DNA replication. This switch is mediated at least in part by changes in transcription elongation and polyadenylation in the late region, and by the formation and editing of dsRNA in the nucleus. In this review we will summarize the regulation of RNA synthesis and processing during polyoma infection, and will point out in particular those aspects that have been most novel.
Assuntos
Polyomavirus/metabolismo , RNA Viral/metabolismo , Animais , Sequência de Bases , DNA Viral/genética , Regulação Viral da Expressão Gênica , Genoma Viral , Camundongos , Dados de Sequência Molecular , Polyomavirus/genética , Polyomavirus/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genéticaRESUMO
We show here that expression of genes from convergent transcription units can be regulated by the formation of double-stranded RNA (dsRNA) in the region of overlapping polyadenylation signals. The model system employed is the mouse polyomavirus. The early and late genes of polyomavirus are transcribed from opposite strands of the circular viral genome. At early times after infection, the early genes are expressed predominantly. Late gene expression increases dramatically upon the onset of DNA replication, when a major defect in polyadenylation of the late primary transcripts generates multigenomic RNAs that are precursors to the mature late mRNAs. Embedded in these late pre-mRNAs are sequences complementary to the early RNAs that act to down-regulate early gene expression via A-to-I editing of dsRNAs. In this system, the defective polyadenylation, and consequently the production of multigenomic late RNAs, depends on the context, and perhaps also, on the A-to-I editing of the poly(A) signal that overlaps the 3'-end of early transcripts.
Assuntos
Regulação da Expressão Gênica , Poliadenilação , RNA Antissenso/metabolismo , RNA de Cadeia Dupla/metabolismo , Animais , Sequência de Bases , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Células NIH 3T3 , Poli A/metabolismo , RNA Antissenso/genética , RNA Mensageiro/metabolismoRESUMO
Vigilin is an RNA-binding protein localized to both the cytoplasm and the nucleus and has been previously implicated in heterochromatin formation and chromosome segregation. We demonstrate here that the C-terminal domain of human vigilin binds to the histone methyltransferase SUV39H1 in vivo. This association is independent of RNA and maps to a site on vigilin that is not involved in its interaction with several other known protein partners. Cells that express high levels of the C-terminal fragment display chromosome segregation defects, and ChIP analyses show changes in the status of pericentric beta-satellite and rDNA chromatin from heterochromatic to more euchromatic form. Finally, a cell line with inducible expression of the vigilin C-terminal fragment displays inducible alterations in beta-satellite chromatin. These and other results lead us to present a new model for vigilin-mediated, RNA-induced gene silencing.
Assuntos
Inativação Gênica , Heterocromatina/metabolismo , Metiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Antígenos Nucleares/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Segregação de Cromossomos/genética , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku , Proteínas de Neoplasias/metabolismo , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Recent work reveals that adenosine-to-inosine editing occurs in a number of cellular microRNAs (miRNAs). Such editing is shown to diminish the expression of one miRNA and alter the target specificity of another. This changes our current views significantly by not only increasing the repertoire of miRNAs and their potential targets, but also providing mechanisms for how to regulate them and direct them to alternative targets.
Assuntos
Adenosina Desaminase/fisiologia , MicroRNAs/fisiologia , Inativação Gênica , Edição de RNA/fisiologia , Interferência de RNA , Proteínas de Ligação a RNARESUMO
Double-stranded RNA (dsRNA) is often formed in the nuclei of mammalian cells, but in this compartment it does not induce the effects characteristic of cytoplasmic dsRNA. Rather, recent work has suggested that nuclear dsRNA is a target for the ADAR class of enzymes, which deaminate adenosines to inosines. Further, there are a number of distinct fates of such edited RNA, including nuclear retention and perhaps also gene silencing.
Assuntos
Núcleo Celular/metabolismo , Edição de RNA , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Adenosina Desaminase/metabolismo , Animais , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , RNA Helicases DEAD-box , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku , Modelos Biológicos , Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , RNA Helicases/metabolismo , Transporte de RNA/fisiologia , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Recent bioinformatic analyses suggest that almost all human transcripts are edited by adenosine deaminases (ADARs), converting adenosines to inosines. Most of this editing is in Alu element transcripts, which are unique to primates. This editing might have no function or might be involved in functions such as the regulation of splicing, chromatin or nuclear localization of transcripts.
Assuntos
Genoma Humano/genética , Edição de RNA/genética , Elementos Nucleotídeos Curtos e Dispersos/genética , Adenosina Desaminase/metabolismo , Elementos Alu/genética , HumanosRESUMO
The fate of double-stranded RNA (dsRNA) in the cell depends on both its length and location . The expression of dsRNA in the nucleus leads to several distinct consequences. First, the promiscuous deamination of adenosines to inosines by dsRNA-specific adenosine deaminase (ADAR) can lead to the nuclear retention of edited transcripts . Second, dsRNAs might induce heterochromatic gene silencing through an RNAi-related mechanism . Is RNA editing also connected to heterochromatin? We report that members of the conserved Vigilin class of proteins have a high affinity for inosine-containing RNAs. In agreement with other work , we find that these proteins localize to heterochromatin and that mutation or depletion of the Drosophila Vigilin, DDP1, leads to altered nuclear morphology and defects in heterochromatin and chromosome segregation. Furthermore, nuclear Vigilin is found in complexes containing not only the editing enzyme ADAR1 but also RNA helicase A and Ku86/70. In the presence of RNA, the Vigilin complex recruits the DNA-PKcs enzyme, which appears to phosphorylate a discrete set of targets, some or all of which are known to participate in chromatin silencing. These results are consistent with a mechanistic link between components of the DNA-repair machinery and RNA-mediated gene silencing.
Assuntos
Proteínas de Transporte/metabolismo , Reparo do DNA , Inativação Gênica , Heterocromatina/metabolismo , Inosina/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Cromatografia de Afinidade , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Citometria de Fluxo , Células HeLa , Humanos , Espectrometria de Massas , Proteínas Nucleares/metabolismo , Plasmídeos/genéticaRESUMO
In this work we describe methods for the analysis of RNAs that have been edited by the double-strand RNA-specific adenosine deaminase, ADAR. These RNAs contain inosine residues that can be detected and quantified by a variety of approaches, including base hydrolysis and thin-layer chromatography, reverse transcription polymerase chain reaction, primer extension, and inosine-specific base cleavage. The most common method for the analysis of editing will be described here. This method involves complete hydrolysis of edited RNAs to nucleoside monophosphates, followed by separation of the products using thin-layer chromatography.