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1.
Mol Cell ; 70(1): 175-187.e8, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29576526

RESUMO

Upon stress, cytoplasmic mRNA is sequestered to insoluble ribonucleoprotein (RNP) granules, such as the stress granule (SG). Partially due to the belief that translationally suppressed mRNAs are recruited to SGs in bulk, stress-induced dynamic redistribution of mRNA has not been thoroughly characterized. Here, we report that endoplasmic reticulum (ER) stress targets only a small subset of translationally suppressed mRNAs into the insoluble RNP granule fraction (RG). This subset, characterized by extended length and adenylate-uridylate (AU)-rich motifs, is highly enriched with genes critical for cell survival and proliferation. This pattern of RG targeting was conserved for two other stress types, heat shock and arsenite toxicity, which induce distinct responses in the total cytoplasmic transcriptome. Nevertheless, stress-specific RG-targeting motifs, such as guanylate-cytidylate (GC)-rich motifs in heat shock, were also identified. Previously underappreciated, transcriptome profiling in the RG may contribute to understanding human diseases associated with RNP dysfunction, such as cancer and neurodegeneration.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Estresse do Retículo Endoplasmático , Resposta ao Choque Térmico , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Transcriptoma , Elementos Ricos em Adenilato e Uridilato , Animais , Arsenitos/toxicidade , Sítios de Ligação , Grânulos Citoplasmáticos/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HCT116 , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Ligação Proteica , Proto-Oncogenes , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Solubilidade , Tapsigargina/toxicidade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/efeitos dos fármacos
2.
Nature ; 571(7765): 424-428, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292544

RESUMO

N6-methyladenosine (m6A) is the most prevalent modified nucleotide in mRNA1,2, with around 25% of mRNAs containing at least one m6A. Methylation of mRNA to form m6A is required for diverse cellular and physiological processes3. Although the presence of m6A in an mRNA can affect its fate in different ways, it is unclear how m6A directs this process and why the effects of m6A can vary in different cellular contexts. Here we show that the cytosolic m6A-binding proteins-YTHDF1, YTHDF2 and YTHDF3-undergo liquid-liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m6A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m6A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m6A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m6A-modified mRNAs are governed by liquid-liquid phase separation principles.


Assuntos
Adenosina/análogos & derivados , Compartimento Celular , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Humanos , Metilação , Metiltransferases/deficiência , Camundongos , Transição de Fase , RNA Mensageiro/análise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico
3.
RNA ; 28(7): 947-971, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35512831

RESUMO

The poly(A) tail enhances translation and transcript stability, and tail length is under dynamic control during cell state transitions. Tail regulation plays essential roles in translational timing and fertilization in early development, but poly(A) tail dynamics have not been fully explored in post-embryonic systems. Here, we examined the landscape and impact of tail length control during macrophage activation. Upon activation, more than 1500 mRNAs, including proinflammatory genes, underwent distinctive changes in tail lengths. Increases in tail length correlated with mRNA levels regardless of transcriptional activity, and many mRNAs that underwent tail extension encode proteins necessary for immune function and post-transcriptional regulation. Strikingly, we found that ZFP36, whose protein product destabilizes target transcripts, undergoes tail extension. Our analyses indicate that many mRNAs undergoing tail lengthening are, in turn, degraded by elevated levels of ZFP36, constituting a post-transcriptional feedback loop that ensures transient regulation of transcripts integral to macrophage activation. Taken together, this study establishes the complexity, relevance, and widespread nature of poly(A) tail dynamics, and the resulting post-transcriptional regulation during macrophage activation.


Assuntos
Ativação de Macrófagos , Poli A , Regulação da Expressão Gênica , Ativação de Macrófagos/genética , Poli A/genética , Poli A/metabolismo , Poliadenilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Nature ; 556(7701): 370-375, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29643508

RESUMO

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding. However, the evolutionary mechanisms that drive cortical size and structure are unknown. Although genes that are essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (a disorder associated with reduced brain size and intellectual disability) 1 , studies of these genes in mice, which have a smooth cortex that is one thousand times smaller than the cortex of humans, have provided limited insight. Mutations in abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by at least 50% in humans2-4, but have little effect on the brains of mice5-9; this probably reflects evolutionarily divergent functions of ASPM10,11. Here we used genome editing to create a germline knockout of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell diversity12-14 than mice, and closer protein sequence homology to the human ASPM protein. Aspm knockout ferrets exhibit severe microcephaly (25-40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as has been found in human patients3,4, suggesting that loss of 'cortical units' has occurred. The cortex of fetal Aspm knockout ferrets displays a very large premature displacement of ventricular radial glial cells to the outer subventricular zone, where many resemble outer radial glia, a subtype of neural progenitor cells that are essentially absent in mice and have been implicated in cerebral cortical expansion in primates12-16. These data suggest an evolutionary mechanism by which ASPM regulates cortical expansion by controlling the affinity of ventricular radial glial cells for the ventricular surface, thus modulating the ratio of ventricular radial glial cells, the most undifferentiated cell type, to outer radial glia, a more differentiated progenitor.


Assuntos
Evolução Biológica , Córtex Cerebral/anatomia & histologia , Córtex Cerebral/metabolismo , Furões , Deleção de Genes , Microcefalia/genética , Microcefalia/patologia , Proteínas do Tecido Nervoso/deficiência , Sequência de Aminoácidos , Animais , Proteínas de Ligação a Calmodulina/deficiência , Proteínas de Ligação a Calmodulina/metabolismo , Centrossomo/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Feminino , Furões/anatomia & histologia , Furões/genética , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Mutação em Linhagem Germinativa , Humanos , Masculino , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Tamanho do Órgão , Transcrição Gênica
5.
Genes Dev ; 28(1): 14-9, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24395245

RESUMO

The kinetics with which promoter-proximal paused RNA polymerase II (Pol II) undergoes premature termination versus productive elongation is central to understanding underlying mechanisms of metazoan transcription regulation. To assess the fate of Pol II quantitatively, we tracked photoactivatable GFP-tagged Pol II at uninduced Hsp70 on polytene chromosomes and showed that Pol II is stably paused with a half-life of 5 min. Biochemical analysis of short nascent RNA from Hsp70 reveals that this half-life is determined by two comparable rates of productive elongation and premature termination of paused Pol II. Importantly, heat shock dramatically increases elongating Pol II without decreasing termination, indicating that regulation acts at the step of paused Pol II entry to productive elongation.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Regiões Promotoras Genéticas/fisiologia , RNA Polimerase II/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Choque Térmico HSP70/genética , Cinética , RNA Polimerase II/genética , Transgenes
6.
Annu Rev Genet ; 47: 483-508, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24050178

RESUMO

Elongation is becoming increasingly recognized as a critical step in eukaryotic transcriptional regulation. Although traditional genetic and biochemical studies have identified major players of transcriptional elongation, our understanding of the importance and roles of these factors is evolving rapidly through the recent advances in genome-wide and single-molecule technologies. Here, we focus on how elongation can modulate the transcriptional outcome through the rate-liming step of RNA polymerase II (Pol II) pausing near promoters and how the participating factors were identified. Among the factors we describe are the pausing factors--NELF (negative elongation factor) and DSIF (DRB sensitivity-inducing factor)--and P-TEFb (positive elongation factor b), which is the key player in pause release. We also describe the high-resolution view of Pol II pausing and propose nonexclusive models for how pausing is achieved. We then discuss Pol II elongation through the bodies of genes and the roles of FACT and SPT6, factors that allow Pol II to move through nucleosomes.


Assuntos
Elongação da Transcrição Genética/fisiologia , Animais , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Células Eucarióticas/metabolismo , Proteínas de Grupo de Alta Mobilidade/fisiologia , Humanos , Mamíferos/genética , Modelos Genéticos , Nucleossomos/genética , Fosforilação , Células Procarióticas/metabolismo , Regiões Promotoras Genéticas/genética , Mapeamento de Interação de Proteínas , Capuzes de RNA/genética , RNA Polimerase II/genética , Splicing de RNA , Ribossomos/genética , Fatores de Transcrição/fisiologia , Fatores de Elongação da Transcrição/fisiologia
7.
Genome Res ; 26(11): 1544-1554, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27638543

RESUMO

Alterations of RNA sequences and structures, such as those from editing and alternative splicing, result in two or more RNA transcripts from a DNA template. It was thought that in yeast, RNA editing only occurs in tRNAs. Here, we found that Saccharomyces cerevisiae have all 12 types of RNA-DNA sequence differences (RDDs) in the mRNA. We showed these sequence differences are propagated to proteins, as we identified peptides encoded by the RNA sequences in addition to those by the DNA sequences at RDD sites. RDDs are significantly enriched at regions with R-loops. A screen of yeast mutants showed that RDD formation is affected by mutations in genes regulating R-loops. Loss-of-function mutations in ribonuclease H, senataxin, and topoisomerase I that resolve RNA-DNA hybrids lead to increases in RDD frequency. Our results demonstrate that RDD is a conserved process that diversifies transcriptomes and proteomes and provide a mechanistic link between R-loops and RDDs.


Assuntos
Pareamento Incorreto de Bases , DNA Fúngico/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , DNA Topoisomerases Tipo I/genética , DNA Fúngico/química , Mutação com Perda de Função , RNA Fúngico/química , RNA Mensageiro/química , Ribonuclease H/genética , Proteínas de Saccharomyces cerevisiae/genética
8.
PLoS Genet ; 10(3): e1004240, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24651406

RESUMO

Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes Hmr and Lhr genetically interact to cause hybrid lethality between Drosophila melanogaster and D. simulans, yet mutations in both genes are viable. Here, we report that Hmr and Lhr encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses we discovered that Hmr and Lhr are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing Hmr and Lhr function between D. melanogaster and D. simulans we identify several satellite DNAs and TEs that are differentially regulated between the species. Hmr and Lhr mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. Hmr and Lhr therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between D. melanogaster and D. simulans and have high potential to cause genetic conflicts with host fitness. We further find that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with Hmr function. Our results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background.


Assuntos
Proteínas de Drosophila/genética , Heterocromatina/genética , Isolamento Reprodutivo , Animais , Evolução Biológica , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Elementos de DNA Transponíveis/genética , DNA Satélite/genética , Drosophila melanogaster , Genes Letais , Hibridização Genética
9.
PLoS Genet ; 10(1): e1004090, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24453987

RESUMO

The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.


Assuntos
HIV-1/genética , Motivos de Nucleotídeos/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases/genética , Drosophila melanogaster/genética , Infecções por HIV/genética , HIV-1/metabolismo , Humanos , Regiões Promotoras Genéticas , RNA/genética , RNA/metabolismo , RNA Polimerase II/genética , Transcrição Gênica
10.
PLoS Genet ; 9(3): e1003382, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23555293

RESUMO

Cohesin is a well-known mediator of sister chromatid cohesion, but it also influences gene expression and development. These non-canonical roles of cohesin are not well understood, but are vital: gene expression and development are altered by modest changes in cohesin function that do not disrupt chromatid cohesion. To clarify cohesin's roles in transcription, we measured how cohesin controls RNA polymerase II (Pol II) activity by genome-wide chromatin immunoprecipitation and precision global run-on sequencing. On average, cohesin-binding genes have more transcriptionally active Pol II and promoter-proximal Pol II pausing than non-binding genes, and are more efficient, producing higher steady state levels of mRNA per transcribing Pol II complex. Cohesin depletion frequently decreases gene body transcription but increases pausing at cohesin-binding genes, indicating that cohesin often facilitates transition of paused Pol II to elongation. In many cases, this likely reflects a role for cohesin in transcriptional enhancer function. Strikingly, more than 95% of predicted extragenic enhancers bind cohesin, and cohesin depletion can reduce their association with Pol II, indicating that cohesin facilitates enhancer-promoter contact. Cohesin depletion decreases the levels of transcriptionally engaged Pol II at the promoters of most genes that don't bind cohesin, suggesting that cohesin controls expression of one or more broadly acting general transcription factors. The multiple transcriptional roles of cohesin revealed by these studies likely underlie the growth and developmental deficits caused by minor changes in cohesin activity.


Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Regiões Promotoras Genéticas , RNA Polimerase II , Animais , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/microbiologia , Regulação da Expressão Gênica no Desenvolvimento , Genoma de Inseto , Histonas/genética , Histonas/metabolismo , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Coesinas
11.
Res Sq ; 2023 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-36909657

RESUMO

Enhancer RNAs (eRNAs) are non-coding RNAs produced from transcriptional enhancers that are highly correlated with their activities. Using capped nascent RNA sequencing (PRO-cap) dataset in human lymphoblastoid cell lines across individuals, we identified inter-individual variation of expression in over 80 thousand transcribed transcriptional regulatory elements (tTREs), in both enhancers and promoters. Co-expression analysis of eRNAs from tTREs across individuals revealed how enhancers interact with each other and with promoters. Mid-to-long range interactions showed distance-dependent decay, which was modified by TF occupancy. In particular, we found a class of 'bivalent' TFs, including Cohesin, which both facilitates and insulates the interaction between enhancers and/or promoters depending on the topology. In short ranges, we observed strand specific interactions between nearby eRNAs in both convergent or divergent orientations. Our finding supports a cooperative convergent eRNA model, which is compatible with eRNA remodeling neighboring enhancers rather than interfering with each other. Therefore, our approach to infer functional interactions from co-expression analyses provided novel insights into the principles of enhancer interactions depending on the distance, orientation, and the binding landscapes of TFs.

12.
Sci Rep ; 13(1): 19085, 2023 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-37925545

RESUMO

Enhancer RNAs (eRNAs) are non-coding RNAs produced by transcriptional enhancers that are highly correlated with their activity. Using a capped nascent RNA sequencing (PRO-cap) dataset in human lymphoblastoid cell lines across 67 individuals, we identified inter-individual variation in the expression of over 80 thousand transcribed transcriptional regulatory elements (tTREs), in both enhancers and promoters. Co-expression analysis of eRNAs from tTREs across individuals revealed how enhancers are associated with each other and with promoters. Mid- to long-range co-expression showed a distance-dependent decay that was modified by TF occupancy. In particular, we found a class of "bivalent" TFs, including Cohesin, that both facilitate and isolate the interaction between enhancers and/or promoters, depending on their topology. At short distances, we observed strand-specific correlations between nearby eRNAs in both convergent and divergent orientations. Our results support a cooperative model of convergent eRNAs, consistent with eRNAs facilitating adjacent enhancers rather than interfering with each other. Therefore, our approach to infer functional interactions from co-expression analyses provided novel insights into the principles of enhancer interactions as a function of distance, orientation, and binding landscapes of TFs.


Assuntos
Elementos Facilitadores Genéticos , Transcrição Gênica , Humanos , RNA/genética , Elementos Reguladores de Transcrição , Regiões Promotoras Genéticas
13.
Cell Mol Immunol ; 20(1): 94-109, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36513810

RESUMO

Monocyte/macrophage lineage cells are highly plastic and can differentiate into various cells under different environmental stimuli. Bone-resorbing osteoclasts are derived from the monocyte/macrophage lineage in response to receptor activator of NF-κB ligand (RANKL). However, the epigenetic signature contributing to the fate commitment of monocyte/macrophage lineage differentiation into human osteoclasts is largely unknown. In this study, we identified RANKL-responsive human osteoclast-specific superenhancers (SEs) and SE-associated enhancer RNAs (SE-eRNAs) by integrating data obtained from ChIP-seq, ATAC-seq, nuclear RNA-seq and PRO-seq analyses. RANKL induced the formation of 200 SEs, which are large clusters of enhancers, while suppressing 148 SEs in macrophages. RANKL-responsive SEs were strongly correlated with genes in the osteoclastogenic program and were selectively increased in human osteoclasts but marginally presented in osteoblasts, CD4+ T cells, and CD34+ cells. In addition to the major transcription factors identified in osteoclasts, we found that BATF binding motifs were highly enriched in RANKL-responsive SEs. The depletion of BATF1/3 inhibited RANKL-induced osteoclast differentiation. Furthermore, we found increased chromatin accessibility in SE regions, where RNA polymerase II was significantly recruited to induce the extragenic transcription of SE-eRNAs, in human osteoclasts. Knocking down SE-eRNAs in the vicinity of the NFATc1 gene diminished the expression of NFATc1, a major regulator of osteoclasts, and osteoclast differentiation. Inhibiting BET proteins suppressed the formation of some RANKL-responsive SEs and NFATc1-associated SEs, and the expression of SE-eRNA:NFATc1. Moreover, SE-eRNA:NFATc1 was highly expressed in the synovial macrophages of rheumatoid arthritis patients exhibiting high-osteoclastogenic potential. Our genome-wide analysis revealed RANKL-inducible SEs and SE-eRNAs as osteoclast-specific signatures, which may contribute to the development of osteoclast-specific therapeutic interventions.


Assuntos
Células da Medula Óssea , Osteoclastos , Ligante RANK , Humanos , Células da Medula Óssea/metabolismo , Diferenciação Celular , Epigênese Genética , Macrófagos/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo
14.
Methods Mol Biol ; 2404: 281-298, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694615

RESUMO

Polyadenylation and deadenylation of mRNA are major RNA modifications associated with nucleus-to-cytoplasm translocation, mRNA stability, translation efficiency, and mRNA decay pathways. Our current knowledge of polyadenylation and deadenylation has been expanded due to recent advances in transcriptome-wide poly(A) tail length assays. Whereas these methods measure poly(A) length by quantifying the adenine (A) base stretch at the 3' end of mRNA, we developed a more cost-efficient technique that does not rely on A-base counting, called tail-end-displacement sequencing (TED-seq). Through sequencing highly size-selected 3' RNA fragments including the poly(A) tail pieces, TED-seq provides accurate measure of transcriptome-wide poly(A)-tail lengths in high resolution, economically suitable for larger scale analysis under various biologically transitional contexts.


Assuntos
Poliadenilação , Genoma , Poli A/genética , Poli A/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
15.
Nat Commun ; 11(1): 5963, 2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-33235186

RESUMO

Enhancer RNAs (eRNA) are unstable non-coding RNAs, transcribed bidirectionally from active regulatory sequences, whose expression levels correlate with enhancer activity. We use capped-nascent-RNA sequencing to efficiently capture bidirectional transcription initiation across several human lymphoblastoid cell lines (Yoruba population) and detect ~75,000 eRNA transcription sites with high sensitivity and specificity. The use of nascent-RNA sequencing sidesteps the confounding effect of eRNA instability. We identify quantitative trait loci (QTLs) associated with the level and directionality of eRNA expression. High-resolution analyses of these two types of QTLs reveal distinct positions of enrichment at the central transcription factor (TF) binding regions and at the flanking eRNA initiation regions, both of which are associated with mRNA expression QTLs. These two regions-the central TF-binding footprint and the eRNA initiation cores-define a bipartite architecture of enhancers, inform enhancer function, and can be used as an indicator of the significance of non-coding regulatory variants.


Assuntos
Elementos Facilitadores Genéticos/genética , Locos de Características Quantitativas , RNA não Traduzido/genética , Linhagem Celular , Regulação da Expressão Gênica/genética , Humanos , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Ribonucleico , Transcrição Gênica
16.
Cell Rep ; 32(8): 108077, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32846134

RESUMO

DNA damage often induces heterogeneous cell-fate responses, such as cell-cycle arrest and apoptosis. Through single-cell RNA sequencing (scRNA-seq), we characterize the transcriptome response of cultured colon cancer cell lines to 5-fluorouracil (5FU)-induced DNA damage. After 5FU treatment, a single population of colon cancer cells adopts three distinct transcriptome phenotypes, which correspond to diversified cell-fate responses: apoptosis, cell-cycle checkpoint, and stress resistance. Although some genes are regulated uniformly across all groups of cells, many genes showed group-specific expression patterns mediating DNA damage responses specific to the corresponding cell fate. Some of these observations are reproduced at the protein level by flow cytometry and are replicated in cells treated with other 5FU-unrelated genotoxic drugs, camptothecin and etoposide. This work provides a resource for understanding heterogeneous DNA damage responses involving fractional killing and chemoresistance, which are among the major challenges in current cancer chemotherapy.


Assuntos
Neoplasias do Colo/genética , Dano ao DNA/genética , Fluoruracila/metabolismo , Análise de Célula Única/métodos , Transcriptoma/genética , Humanos
17.
Cell Rep ; 24(13): 3630-3641.e7, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30257221

RESUMO

Post-transcriptional RNA processing is a core mechanism of gene expression control in cell stress response. The poly(A) tail influences mRNA translation and stability, but it is unclear whether there are global roles of poly(A)-tail lengths in cell stress. To address this, we developed tail-end displacement sequencing (TED-seq) for an efficient transcriptome-wide profiling of poly(A) lengths and applied it to endoplasmic reticulum (ER) stress in human cells. ER stress induced increases in the poly(A) lengths of certain mRNAs, including known ER stress regulators, XBP1, DDIT3, and HSPA5. Importantly, the mRNAs with increased poly(A) lengths are both translationally de-repressed and stabilized. Furthermore, mRNAs in stress-induced RNA granules have shorter poly(A) tails than in the cytoplasm, supporting the view that RNA processing is compartmentalized. In conclusion, TED-seq reveals that poly(A) length is dynamically regulated upon ER stress, with potential consequences for both translation and mRNA turnover.


Assuntos
Estresse do Retículo Endoplasmático , Poli A/metabolismo , Poliadenilação , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Poli A/química , Análise de Sequência de RNA/métodos , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Transcriptoma , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
18.
Nat Genet ; 50(11): 1553-1564, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30349114

RESUMO

The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases.


Assuntos
Neoplasias Encefálicas/genética , Mapeamento Cromossômico/métodos , Glioblastoma/genética , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Cromatina/genética , Cromatina/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano , Glioblastoma/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Células Jurkat , Desequilíbrio de Ligação , Camundongos , Camundongos Nus , Elongação da Transcrição Genética
19.
Nat Protoc ; 11(8): 1455-76, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27442863

RESUMO

We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3' end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3' end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4-5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.


Assuntos
Pareamento de Bases , Mapeamento Cromossômico/métodos , RNA Polimerases Dirigidas por DNA/metabolismo , RNA/química , RNA/genética , Animais , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Humanos , Camundongos , RNA/metabolismo , Análise de Sequência de RNA , Sítio de Iniciação de Transcrição
20.
Cell Rep ; 16(7): 2003-16, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27498870

RESUMO

Bromodomain and extra-terminal domain (BET) family inhibitors offer an approach to treating hematological malignancies. We used precision nuclear run-on transcription sequencing (PRO-seq) to create high-resolution maps of active RNA polymerases across the genome in t(8;21) acute myeloid leukemia (AML), as these polymerases are exceptionally sensitive to BET inhibitors. PRO-seq identified over 1,400 genes showing impaired release of promoter-proximal paused RNA polymerases, including the stem cell factor receptor tyrosine kinase KIT that is mutated in t(8;21) AML. PRO-seq also identified an enhancer 3' to KIT. Chromosome conformation capture confirmed contacts between this enhancer and the KIT promoter, while CRISPRi-mediated repression of this enhancer impaired cell growth. PRO-seq also identified microRNAs, including MIR29C and MIR29B2, that target the anti-apoptotic factor MCL1 and were repressed by BET inhibitors. MCL1 protein was upregulated, and inhibition of BET proteins sensitized t(8:21)-containing cells to MCL1 inhibition, suggesting a potential mechanism of resistance to BET-inhibitor-induced cell death.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Translocação Genética , Antineoplásicos/farmacologia , Azepinas/farmacologia , Linhagem Celular Tumoral , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , RNA Polimerases Dirigidas por DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Elementos Facilitadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Família Multigênica , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transcrição Gênica , Triazóis/farmacologia
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