RESUMO
Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3'-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field.
Assuntos
Precursores de RNA , Fatores de Poliadenilação e Clivagem de mRNA , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Expressão GênicaRESUMO
In eukaryotes, poly(A) tails are present on almost every mRNA. Early experiments led to the hypothesis that poly(A) tails and the cytoplasmic polyadenylate-binding protein (PABPC) promote translation and prevent mRNA degradation, but the details remained unclear. More recent data suggest that the role of poly(A) tails is much more complex: poly(A)-binding protein can stimulate poly(A) tail removal (deadenylation) and the poly(A) tails of stable, highly translated mRNAs at steady state are much shorter than expected. Furthermore, the rate of translation elongation affects deadenylation. Consequently, the interplay between poly(A) tails, PABPC, translation and mRNA decay has a major role in gene regulation. In this Review, we discuss recent work that is revolutionizing our understanding of the roles of poly(A) tails in the cytoplasm. Specifically, we discuss the roles of poly(A) tails in translation and control of mRNA stability and how poly(A) tails are removed by exonucleases (deadenylases), including CCR4-NOT and PAN2-PAN3. We also discuss how deadenylation rate is determined, the integration of deadenylation with other cellular processes and the function of PABPC. We conclude with an outlook for the future of research in this field.
Assuntos
Eucariotos/genética , Regulação da Expressão Gênica , Poli A/metabolismo , RNA Mensageiro/metabolismo , Animais , Humanos , Biossíntese de Proteínas/genética , Estabilidade de RNA , RNA Mensageiro/genéticaRESUMO
The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3' end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cromatina , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Proteínas de Domínio MADS , RNA Polimerase II , Terminação da Transcrição Genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimologia , Cromatina/metabolismo , Cromatina/genética , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Histonas/metabolismo , Histonas/genética , Histona DesacetilasesRESUMO
To celebrate the 50th anniversary of Cell Press and the Cell focus issue on structural biology, we discussed with scientists working across diverse fields how AlphaFold has changed their research and brought structural biology to the masses.
Assuntos
Aniversários e Eventos Especiais , Biologia MolecularRESUMO
Gene expression is controlled in a dynamic and regulated manner to allow for the consistent and steady expression of some proteins as well as the rapidly changing production of other proteins. Transcription initiation has been a major focus of study because it is highly regulated. However, termination of transcription also plays an important role in controlling gene expression. Transcription termination on protein-coding genes is intimately linked with 3' end cleavage and polyadenylation of transcripts, and it generally results in the production of a mature mRNA that is exported from the nucleus. Termination on many non-coding genes can also result in the production of a mature transcript. Termination is dynamically regulated-premature termination and transcription readthrough occur in response to a number of cellular signals, and these can have varied consequences on gene expression. Here, we review eukaryotic transcription termination by RNA polymerase II (RNAPII), focusing on protein-coding genes.
Assuntos
RNA Polimerase II , Transcrição Gênica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Poliadenilação , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terminação da Transcrição GenéticaRESUMO
Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, and protein phosphatase activities, which cleave and polyadenylate pre-mRNAs and dephosphorylate RNA Pol II to control transcription. Exactly how the RNA 3' end processing machinery is coupled to transcription remains unclear. Here, we combine in vitro reconstitution, structural studies, and genome-wide analyses to show that yeast CPF physically and functionally interacts with RNA Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of an RNA Pol II stalk-to-stalk homodimer in vitro. This dimer is compatible with transcription but not with the binding of transcription elongation factors. Disruption of the dimerization interface in cells causes transcription defects, including altered RNA Pol II abundance on protein-coding genes, tRNA genes, and intergenic regions. We hypothesize that RNA Pol II dimerization may provide a mechanistic basis for the allosteric model of transcription termination.
Assuntos
RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estudo de Associação Genômica Ampla , Transcrição Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Processamento de Terminações 3' de RNA/genéticaRESUMO
Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.
Assuntos
Ribossomos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Microtúbulos/metabolismo , Homeostase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estabilidade de RNA , Proteínas de Transporte/metabolismo , Fatores de Transcrição/metabolismoRESUMO
DNA crosslinks block DNA replication and are repaired by the Fanconi anaemia pathway. The FANCD2-FANCI (D2-I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion1. However, D2-I is also known to have a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation2-4. At present, it is unclear how DNA crosslinks are recognized and how D2-I functions in replication fork protection. Here, using single-molecule imaging, we show that D2-I is a sliding clamp that binds to and diffuses on double-stranded DNA. Notably, sliding D2-I stalls on encountering single-stranded-double-stranded (ss-ds) DNA junctions, structures that are generated when replication forks stall at DNA lesions5. Using cryogenic electron microscopy, we determined structures of D2-I on DNA that show that stalled D2-I makes specific interactions with the ss-dsDNA junction that are distinct from those made by sliding D2-I. Thus, D2-I surveys dsDNA and, when it reaches an ssDNA gap, it specifically clamps onto ss-dsDNA junctions. Because ss-dsDNA junctions are found at stalled replication forks, D2-I can identify sites of DNA damage. Therefore, our data provide a unified molecular mechanism that reconciles the roles of D2-I in the recognition and protection of stalled replication forks in several DNA repair pathways.
RESUMO
Most eukaryotic messenger RNAs (mRNAs) are processed at their 3' end by the cleavage and polyadenylation specificity factor (CPF/CPSF). CPF mediates the endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3' end of the mature transcript. The activation of CPF is highly regulated to maintain the fidelity of RNA processing. Here, using cryo-EM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre-mRNA. The region of Mpe1 that contacts RNA also promotes the activation of CPF endonuclease activity and controls polyadenylation. The Cft2 subunit of CPF antagonizes the RNA-stabilized configuration of Mpe1. In vivo, the depletion or mutation of Mpe1 leads to widespread defects in transcription termination by RNA polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in accurate 3' end processing, activating CPF, and ensuring timely transcription termination.
Assuntos
Precursores de RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Poliadenilação e Clivagem de mRNA , Sequência de Aminoácidos , Microscopia Crioeletrônica , Poliadenilação , Ligação Proteica , Estrutura Terciária de Proteína , Precursores de RNA/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
3' end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3' end of the mature transcript, which is important for mRNA localization, translation, and stability. Cleavage must therefore be tightly regulated. Here, we reconstituted specific and efficient 3' endonuclease activity of human CPSF with purified proteins. This required the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm), and, importantly, the multidomain protein RBBP6. Unlike its yeast homolog Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to humans. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in mRNA processing.
Assuntos
Proteínas de Ligação a DNA , Precursores de RNA , Ubiquitina-Proteína Ligases , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Cleavage and polyadenylation factor (CPF/CPSF) is a multiprotein complex essential for mRNA 3' end processing in eukaryotes. It contains an endonuclease that cleaves pre-mRNAs, and a polymerase that adds a poly(A) tail onto the cleaved 3' end. Several CPF subunits, including Fip1, contain intrinsically disordered regions (IDRs). IDRs within multiprotein complexes can be flexible, or can become ordered upon interaction with binding partners. Here, we show that yeast Fip1 anchors the poly(A) polymerase Pap1 onto CPF via an interaction with zinc finger 4 of another CPF subunit, Yth1. We also reconstitute a fully recombinant 850-kDa CPF. By incorporating selectively labeled Fip1 into recombinant CPF, we could study the dynamics of Fip1 within the megadalton complex using nuclear magnetic resonance (NMR) spectroscopy. This reveals that a Fip1 IDR that connects the Yth1- and Pap1-binding sites remains highly dynamic within CPF. Together, our data suggest that Fip1 dynamics within the 3' end processing machinery are required to coordinate cleavage and polyadenylation.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Poliadenilação , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Biogenesis of most eukaryotic mRNAs involves the addition of an untemplated polyadenosine (pA) tail by the cleavage and polyadenylation machinery. The pA tail, and its exact length, impacts mRNA stability, nuclear export, and translation. To define how polyadenylation is controlled in S. cerevisiae, we have used an in vivo assay capable of assessing nuclear pA tail synthesis, analyzed tail length distributions by direct RNA sequencing, and reconstituted polyadenylation reactions with purified components. This revealed three control mechanisms for pA tail length. First, we found that the pA binding protein (PABP) Nab2p is the primary regulator of pA tail length. Second, when Nab2p is limiting, the nuclear pool of Pab1p, the second major PABP in yeast, controls the process. Third, when both PABPs are absent, the cleavage and polyadenylation factor (CPF) limits pA tail synthesis. Thus, Pab1p and CPF provide fail-safe mechanisms to a primary Nab2p-dependent pathway, thereby preventing uncontrolled polyadenylation and allowing mRNA export and translation.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Poliadenilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3' ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3' end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit "CPFcore" complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPFcore. Together, our data suggest that the active mRNA 3' end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.
Assuntos
Endonucleases/metabolismo , Poliadenilação , Precursores de RNA/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Citocromos c/genética , Citocromos c/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Ativação Enzimática , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Complexos Multiproteicos , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Precursores de RNA/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Translation and decay of eukaryotic mRNAs is controlled by shortening of the poly(A) tail and release of the poly(A)-binding protein Pab1/PABP. The Ccr4-Not complex contains two exonucleases-Ccr4 and Caf1/Pop2-that mediate mRNA deadenylation. Here, using a fully reconstituted biochemical system with proteins from the fission yeast Schizosaccharomyces pombe, we show that Pab1 interacts with Ccr4-Not, stimulates deadenylation, and differentiates the roles of the nuclease enzymes. Surprisingly, Pab1 release relies on Ccr4 activity. In agreement with this, in vivo experiments in budding yeast show that Ccr4 is a general deadenylase that acts on all mRNAs. In contrast, Caf1 only trims poly(A) not bound by Pab1. As a consequence, Caf1 is a specialized deadenylase required for the selective deadenylation of transcripts with lower rates of translation elongation and reduced Pab1 occupancy. These findings reveal a coupling between the rates of translation and deadenylation that is dependent on Pab1 and Ccr4-Not.
Assuntos
Exorribonucleases/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Citoplasma/metabolismo , Endonucleases/metabolismo , Exorribonucleases/genética , Poli A/metabolismo , Poliadenilação , Estabilidade de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonucleases/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair.
Assuntos
Microscopia Crioeletrônica , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Subunidades Proteicas/química , Animais , Galinhas , Anemia de Fanconi/enzimologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/ultraestrutura , Espectrometria de Massas , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Relação Estrutura-Atividade , UbiquitinaçãoRESUMO
We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Dano ao DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hidroxiureia/farmacologia , Proteínas Nucleares/genética , Proteínas de Ligação a Poli-ADP-Ribose , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Fatores de Transcrição/genética , Ubiquitina/genética , UbiquitinaçãoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Leucemia Mieloide Aguda/metabolismo , Precursores de RNA/metabolismo , Sarcoma de Ewing/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Fator de Especificidade de Clivagem e Poliadenilação/genética , Células HEK293 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fenótipo , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Piperazinas/farmacologia , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Sarcoma de Ewing/tratamento farmacológicoRESUMO
Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.
Assuntos
Proteínas Aviárias/metabolismo , Galinhas/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Ubiquitinação , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Linhagem Celular , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C-P lyase core complex (PhnG-PhnH-PhnI-PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.