RESUMO
The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.
Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/genética , Exorribonucleases/genética , Metiltransferases/genética , SARS-CoV-2/genética , Sequência de Aminoácidos , COVID-19/virologia , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Alinhamento de Sequência , Transcrição Gênica/genética , Replicação Viral/genéticaRESUMO
Widespread mRNA decay, an unappreciated feature of apoptosis, enhances cell death and depends on mitochondrial outer membrane permeabilization (MOMP), TUTases, and DIS3L2. Which RNAs are decayed and the decay-initiating event are unknown. Here, we show extensive decay of mRNAs and poly(A) noncoding (nc)RNAs at the 3' end, triggered by the mitochondrial intermembrane space 3'-to-5' exoribonuclease PNPT1, released during MOMP. PNPT1 knockdown inhibits apoptotic RNA decay and reduces apoptosis, while ectopic expression of PNPT1, but not an RNase-deficient mutant, increases RNA decay and cell death. The 3' end of PNPT1 substrates thread through a narrow channel. Many non-poly(A) ncRNAs contain 3'-secondary structures or bind proteins that may block PNPT1 activity. Indeed, mutations that disrupt the 3'-stem-loop of a decay-resistant ncRNA render the transcript susceptible, while adding a 3'-stem-loop to an mRNA prevents its decay. Thus, PNPT1 release from mitochondria during MOMP initiates apoptotic decay of RNAs lacking 3'-structures.
Assuntos
Apoptose , Exorribonucleases/metabolismo , Mitocôndrias/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Citocromos c/metabolismo , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/genética , Células HCT116 , Humanos , Membranas Mitocondriais/metabolismo , Conformação de Ácido Nucleico , Permeabilidade , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/química , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/química , RNA não Traduzido/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in RNA exosome genes are associated with neurodegenerative diseases. Little is known about the role of the RNA exosome in the cellular response to pathogens. Here, using NGS and human and mouse genetics, we show that influenza A virus (IAV) ribogenesis and growth are suppressed by impaired RNA exosome activity. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. The viral polymerase complex co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3' end degradation. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Our results suggest that evolutionary arms races have shaped the cellular RNA quality control machinery.
Assuntos
Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Humana/virologia , RNA Polimerase II/metabolismo , Células A549 , Animais , Imunoprecipitação da Cromatina , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Exossomos/metabolismo , Humanos , Espectrometria de Massas , Camundongos , Mutação , Doenças Neurodegenerativas/virologia , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Transcrição GênicaRESUMO
The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and senataxin) with the noncoding RNA processing function of RNA exosome determine the strand-specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development.
Assuntos
Linfócitos B/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Núcleo Celular/metabolismo , DNA Helicases/metabolismo , Exorribonucleases/genética , Instabilidade Genômica , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Enzimas Multifuncionais , Proteínas Nucleares/genética , RNA Helicases , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genéticaRESUMO
N6-methyladenosine (m6A) is a crucial RNA modification that regulates diverse biological processes in human cells, but its co-transcriptional deposition and functions remain poorly understood. Here, we identified the RNA helicase DDX21 with a previously unrecognized role in directing m6A modification on nascent RNA for co-transcriptional regulation. DDX21 interacts with METTL3 for co-recruitment to chromatin through its recognition of R-loops, which can be formed co-transcriptionally as nascent transcripts hybridize onto the template DNA strand. Moreover, DDX21's helicase activity is needed for METTL3-mediated m6A deposition onto nascent RNA following recruitment. At transcription termination regions, this nexus of actions promotes XRN2-mediated termination of RNAPII transcription. Disruption of any of these steps, including the loss of DDX21, METTL3, or their enzymatic activities, leads to defective termination that can induce DNA damage. Therefore, we propose that the R-loop-DDX21-METTL3 nexus forges the missing link for co-transcriptional modification of m6A, coordinating transcription termination and genome stability.
Assuntos
Adenosina , Adenosina/análogos & derivados , RNA Helicases DEAD-box , Exorribonucleases , Instabilidade Genômica , Metiltransferases , Estruturas R-Loop , RNA Polimerase II , Terminação da Transcrição Genética , Humanos , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Adenosina/metabolismo , Adenosina/genética , Exorribonucleases/metabolismo , Exorribonucleases/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Células HEK293 , Cromatina/metabolismo , Cromatina/genética , Dano ao DNA , Células HeLa , RNA/metabolismo , RNA/genética , Transcrição Gênica , Metilação de RNARESUMO
The superkiller (SKI) complex is the cytoplasmic co-factor and regulator of the RNA-degrading exosome. In human cells, the SKI complex functions mainly in co-translational surveillance-decay pathways, and its malfunction is linked to a severe congenital disorder, the trichohepatoenteric syndrome. To obtain insights into the molecular mechanisms regulating the human SKI (hSKI) complex, we structurally characterized several of its functional states in the context of 80S ribosomes and substrate RNA. In a prehydrolytic ATP form, the hSKI complex exhibits a closed conformation with an inherent gating system that effectively traps the 80S-bound RNA into the hSKI2 helicase subunit. When active, hSKI switches to an open conformation in which the gating is released and the RNA 3' end exits the helicase. The emerging picture is that the gatekeeping mechanism and architectural remodeling of hSKI underpin a regulated RNA channeling system that is mechanistically conserved among the cytoplasmic and nuclear helicase-exosome complexes.
Assuntos
Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Helicases/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA/metabolismo , Subunidades Ribossômicas/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Exorribonucleases/genética , Exorribonucleases/ultraestrutura , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/ultraestrutura , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA/genética , RNA/ultraestrutura , RNA Helicases/genética , RNA Helicases/ultraestrutura , Subunidades Ribossômicas/genética , Subunidades Ribossômicas/ultraestrutura , Relação Estrutura-AtividadeRESUMO
The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.
Assuntos
Núcleo Celular/enzimologia , Proliferação de Células , Replicação do DNA , Exossomos/enzimologia , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/enzimologia , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Quebras de DNA de Cadeia Dupla , Exorribonucleases/genética , Exorribonucleases/metabolismo , Exossomos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Células NIH 3T3 , Neuroblastoma/genética , Neuroblastoma/patologia , Regiões Promotoras Genéticas , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Polimerase II/genética , Terminação da Transcrição GenéticaRESUMO
Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Bacteriano/genética , Domínio Catalítico/genética , Endorribonucleases/genética , Exorribonucleases/genética , Regulação Bacteriana da Expressão Gênica/genética , Chaperonas Moleculares/genética , Estabilidade de RNA/genética , Pequeno RNA não Traduzido/genéticaRESUMO
Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly because of technical limitations in purifying authentic nascent transcripts. We present a new approach to characterize nascent RNA, called polymerase intact nascent transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5' ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano), and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5' P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that co-transcriptional splicing either occurs immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.
Assuntos
Nanotecnologia , RNA Polimerase II/metabolismo , Precursores de RNA/biossíntese , Splicing de RNA , RNA Mensageiro/biossíntese , RNA-Seq , Transcrição Gênica , Exorribonucleases/genética , Exorribonucleases/metabolismo , Células HCT116 , Células HeLa , Humanos , Cinética , Poliadenilação , Capuzes de RNA , RNA Polimerase II/genética , Precursores de RNA/genética , RNA Mensageiro/genéticaRESUMO
Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate1-3. Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA-degradation complex known as the rixosome or RIX1 complex4-6. Whether RNA degradation also has a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5' monophosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosomal degradation of nascent RNA is conserved from fission yeast to human, with a primary role in RNA degradation at facultative heterochromatin in human cells.
Assuntos
Inativação Gênica , Heterocromatina , Complexo Repressor Polycomb 1 , Estabilidade de RNA , Exorribonucleases/genética , Heterocromatina/genética , Humanos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Proteínas do Grupo Polycomb/genética , Schizosaccharomyces/genéticaRESUMO
An attractive approach to reduce gene expression is via the use of antisense oligonucleotides (ASOs) that harness the RNase H1 mechanism. Here we show that RNase H ASOs targeted to introns or exons robustly reduce the level of spliced RNA associated with chromatin. Surprisingly, intron-targeted ASOs reduce the level of pre-mRNA associated with chromatin to a greater extent than exon-targeted ASOs. This indicates that exon-targeted ASOs achieve full activity after the pre-mRNA has undergone splicing, but before the mRNA is released from chromatin. Even though RNase H ASOs can reduce the level of RNA associated with chromatin, the effect of ASO-directed RNA degradation on transcription has never been documented. Here we show that intron-targeted ASOs and, to a lesser extent, exon-targeted ASOs cause RNA polymerase II (Pol II) transcription termination in cultured cells and mice. Furthermore, ASO-directed transcription termination is mediated by the nuclear exonuclease XRN2.
Assuntos
Cromatina/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Terminação da Transcrição Genética , Animais , Cromatina/genética , Éxons , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Células HCT116 , Humanos , Íntrons , Camundongos Endogâmicos C57BL , Modelos Genéticos , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Oligonucleotídeos Antissenso/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Ribonuclease H/genética , Fatores de TempoRESUMO
RNA turnover regulates the quality and quantity of cellular gene expression through a coordinated cavalcade of enzymes, factors, and phase transitions. In this issue, Brothers et al reveal the importance of balanced communication between the Xrn1 exonuclease and the EDC4 decapping factor to coordinate P-body dynamics and maintain cellular fitness.
Assuntos
Endorribonucleases , Corpos de Processamento , Endorribonucleases/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Estabilidade de RNARESUMO
EXD2 is a recently identified exonuclease that cleaves RNA and DNA in double-stranded (ds) forms. It thus serves as a model system for investigating the similarities and discrepancies between exoribonuclease and exodeoxyribonuclease activities and for understanding the nucleic acid (NA) unwinding-degradation coordination of an exonuclease. Here, using a single-molecule fluorescence resonance energy transfer (smFRET) approach, we show that despite stable binding to both substrates, EXD2 barely cleaves dsDNA and yet displays both exoribonuclease and exodeoxyribonuclease activities toward RNA-DNA hybrids with a cleavage preference for RNA. Unexpectedly, EXD2-mediated hybrid cleavage proceeds in a discrete stepwise pattern, wherein a sudden 4-bp duplex unwinding increment and the subsequent dwell constitute a complete hydrolysis cycle. The relatively weak exodeoxyribonuclease activity of EXD2 partially originates from frequent hybrid rewinding. Importantly, kinetic analysis and comparison of the dwell times under varied conditions reveal two rate-limiting steps of hybrid unwinding and nucleotide excision. Overall, our findings help better understand the cellular functions of EXD2, and the cyclic coupling between duplex unwinding and exonucleolytic degradation may be generalizable to other exonucleases.
Assuntos
Exorribonucleases , RNA , RNA/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Cinética , DNA/metabolismo , Exodesoxirribonucleases/metabolismoRESUMO
Deadenylation-dependent mRNA decapping and decay is the major cytoplasmic mRNA turnover pathway in eukaryotes. Many mRNA decapping and decay factors are associated with each other via protein-protein interaction motifs. For example, the decapping enzyme DCP2 and the 5'-3' exonuclease XRN1 interact with the enhancer of mRNA-decapping protein 4 (EDC4), a large scaffold that has been reported to stimulate mRNA decapping. mRNA decapping and decay factors are also found in processing bodies (P-bodies), evolutionarily conserved ribonucleoprotein granules that are often enriched with mRNAs targeted for decay, yet paradoxically are not required for mRNA decay to occur. Here, we show that disrupting the EDC4-XRN1 interaction or altering their stoichiometry inhibits mRNA decapping, with microRNA-targeted mRNAs being stabilized in a translationally repressed state. Importantly, we demonstrate that this concomitantly leads to larger P-bodies that are responsible for preventing mRNA decapping. Finally, we demonstrate that P-bodies support cell viability and prevent stress granule formation when XRN1 is limiting. Taken together, these data demonstrate that the interaction between XRN1 and EDC4 regulates P-body dynamics to properly coordinate mRNA decapping with 5'-3' decay in human cells.
Assuntos
Endorribonucleases , Corpos de Processamento , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas/metabolismo , Eucariotos/genética , Eucariotos/metabolismo , Estabilidade de RNA/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Mutations in RNA-processing enzymes are increasingly linked to human disease. Telomerase RNA and related noncoding RNAs require 3' end-processing steps, including oligoadenylation. Germline mutations in poly(A)ribonuclease (PARN) cause accumulation of extended human telomerase RNA (hTR) species and precipitate dyskeratosis congenita and pulmonary fibrosis. Here, we develop nascent RNAend-seq to measure processing rates of RNA precursors. We find that mature hTR derives from extended precursors but that in PARN-mutant cells hTR maturation kinetically stalls and unprocessed precursors are degraded. Loss of poly(A)polymerase PAPD5 in PARN-mutant cells accelerates hTR maturation and restores hTR processing, indicating that oligoadenylation and deadenylation set rates of hTR maturation. The H/ACA domain mediates hTR maturation by precisely defining the 3' end, recruiting poly(A)polymerase activity, and conferring sensitivity to PARN regulation. These data reveal a feedforward circuit in which post-transcriptional oligoadenylation controls RNA maturation kinetics. Similar alterations in RNA processing rates may contribute to mechanisms of RNA-based human disease.
Assuntos
Disceratose Congênita/genética , Exorribonucleases/genética , RNA Nucleotidiltransferases/genética , RNA/genética , Telomerase/genética , Disceratose Congênita/patologia , Mutação em Linhagem Germinativa/genética , Células HeLa , Humanos , Cinética , Processamento Pós-Transcricional do RNA/genéticaRESUMO
Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.
Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Exorribonucleases/metabolismo , Mitocôndrias/enzimologia , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Mitocondrial/metabolismo , Proteínas 14-3-3/deficiência , Proteínas 14-3-3/genética , Animais , Biomarcadores Tumorais/genética , Exorribonucleases/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mitocondrial/genética , Células Sf9 , SpodopteraRESUMO
Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA polymerase II (Pol II) elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, Pol II decelerates from >2 kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a model for termination, the "sitting duck torpedo" mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert Pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homolog NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/metabolismo , Terminação da Transcrição Genética , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Exorribonucleases/genética , Células HEK293 , Humanos , Cinética , Proteínas Nucleares/genética , Fosforilação , Poli A/metabolismo , Ligação Proteica , Proteína Fosfatase 1/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Elongação da Transcrição/genéticaRESUMO
The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.
Assuntos
Células Epiteliais/virologia , Perfilação da Expressão Gênica/métodos , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Sindbis virus/genética , Transcriptoma , Neoplasias do Colo do Útero/virologia , Regiões 5' não Traduzidas , Sítios de Ligação , Células Epiteliais/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN , Sindbis virus/crescimento & desenvolvimento , Sindbis virus/metabolismo , Sindbis virus/patogenicidade , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Replicação ViralRESUMO
PARN loss-of-function mutations cause a severe form of the hereditary disease dyskeratosis congenita (DC). PARN deficiency affects the stability of non-coding RNAs such as human telomerase RNA (hTR), but these effects do not explain the severe disease in patients. We demonstrate that PARN deficiency affects the levels of numerous miRNAs in human cells. PARN regulates miRNA levels by stabilizing either mature or precursor miRNAs by removing oligo(A) tails added by the poly(A) polymerase PAPD5, which if remaining recruit the exonuclease DIS3L or DIS3L2 to degrade the miRNA. PARN knockdown destabilizes multiple miRNAs that repress p53 translation, which leads to an increase in p53 accumulation in a Dicer-dependent manner, thus explaining why PARN-defective patients show p53 accumulation. This work also reveals that DIS3L and DIS3L2 are critical 3' to 5' exonucleases that regulate miRNA stability, with the addition and removal of 3' end extensions controlling miRNA levels in the cell.
Assuntos
Exorribonucleases/metabolismo , MicroRNAs/metabolismo , Estabilidade de RNA , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/enzimologia , Regiões 3' não Traduzidas , Antineoplásicos/farmacologia , Sobrevivência Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Exorribonucleases/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , MicroRNAs/genética , Poliadenilação , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
ERI1 is a 3'-to-5' exoribonuclease involved in RNA metabolic pathways including 5.8S rRNA processing and turnover of histone mRNAs. Its biological and medical significance remain unclear. Here, we uncover a phenotypic dichotomy associated with bi-allelic ERI1 variants by reporting eight affected individuals from seven unrelated families. A severe spondyloepimetaphyseal dysplasia (SEMD) was identified in five affected individuals with missense variants but not in those with bi-allelic null variants, who showed mild intellectual disability and digital anomalies. The ERI1 missense variants cause a loss of the exoribonuclease activity, leading to defective trimming of the 5.8S rRNA 3' end and a decreased degradation of replication-dependent histone mRNAs. Affected-individual-derived induced pluripotent stem cells (iPSCs) showed impaired in vitro chondrogenesis with downregulation of genes regulating skeletal patterning. Our study establishes an entity previously unreported in OMIM and provides a model showing a more severe effect of missense alleles than null alleles within recessive genotypes, suggesting a key role of ERI1-mediated RNA metabolism in human skeletal patterning and chondrogenesis.