RESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Cromatina , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Proteínas de Dominio MADS , ARN Polimerasa II , Terminación de la Transcripción Genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimología , Cromatina/metabolismo , Cromatina/genética , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Histonas/metabolismo , Histonas/genética , Histona DesacetilasasRESUMEN
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.
Asunto(s)
Aniversarios y Eventos Especiales , Biología MolecularRESUMEN
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.
Asunto(s)
ARN Polimerasa II , Proteínas de Saccharomyces cerevisiae , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estudio de Asociación del Genoma Completo , Transcripción Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Procesamiento de Término de ARN 3'/genéticaRESUMEN
Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4-Not deadenylation complex. The details of this process in mammals remain unclear. Here, we use cryogenic electron microscopy (cryo-EM) and crosslinking mass spectrometry to show that mammalian CCR4-NOT specifically recognizes ribosomes that are stalled during translation elongation in an in vitro reconstituted system with rabbit and human components. Similar to yeast, mammalian CCR4-NOT inserts a helical bundle of its CNOT3 subunit into the empty E site of the ribosome. Our cryo-EM structure shows that CNOT3 also locks the L1 stalk in an open conformation to inhibit further translation. CCR4-NOT is required for stable association of the nonconstitutive subunit CNOT4, which ubiquitinates the ribosome, likely to signal stalled translation elongation. Overall, our work shows that human CCR4-NOT not only detects but also enforces ribosomal stalling to couple translation and mRNA decay.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Animales , Conejos , Mamíferos , Ribosomas , Ubiquitinación , Espectrometría de Masas , Factores de Transcripción , Receptores CCR4 , RibonucleasasRESUMEN
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.
Asunto(s)
Ribosomas , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Microtúbulos/metabolismo , Homeostasis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estabilidad del ARN , Proteínas Portadoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Lori Passmore is a Group Leader at the MRC Laboratory of Molecular Biology (MRC-LMB). She studied Biochemistry at the University of British Columbia in Vancouver (Canada), before moving to the UK in 1999 for a PhD at the Institute of Cancer Research. After completing her PhD, Lori moved to Cambridge, where she became a Post-Doctoral Fellow at the MRC-LMB. In 2009, Lori started her own group at the MRC-LMB and was subsequently awarded an ERC Starting Grant (2011), an ERC Consolidator Grant (2017) and a Wellcome Discovery Award (2023). She was also elected into the EMBO Young Investigator Programme (2015) and EMBO Membership (2018). Lori's research focusses on the determination of the structures of protein complexes that regulate gene expression, using primarily cryo-electron microscopy and in vitro assays. Her work has contributed significantly to our understanding of the underlying molecular mechanisms of cellular processes, giving insights into human physiology and disease. In this interview, Lori provides an overview of her research and discusses current challenges in the field, recalls the key events and collaborations that have helped shape her successful research career and offers advice to early career scientists.
Asunto(s)
Distinciones y Premios , Neoplasias , Femenino , Humanos , Microscopía por Crioelectrón , Biología Molecular , InvestigadoresRESUMEN
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.
Asunto(s)
Precursores del ARN , Factores de Escisión y Poliadenilación de ARNm , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Expresión GénicaRESUMEN
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.
Asunto(s)
ARN Polimerasa II , Transcripción Genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Poliadenilación , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terminación de la Transcripción GenéticaRESUMEN
DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2-FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2-FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2-FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2-FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2-FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2-FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair.
Asunto(s)
Anemia de Fanconi , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , ADN/metabolismo , Daño del ADN , Reparación del ADN , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Humanos , Polinucleótido 5'-Hidroxil-Quinasa/genética , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Precursores del ARN , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factores de Escisión y Poliadenilación de ARNm , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Poliadenilación , Unión Proteica , Estructura Terciaria de Proteína , Precursores del ARN/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN , Precursores del ARN , Ubiquitina-Proteína Ligasas , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Humanos , Precursores del ARN/genética , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
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.
Asunto(s)
Eucariontes/genética , Regulación de la Expresión Génica , Poli A/metabolismo , ARN Mensajero/metabolismo , Animales , Humanos , Biosíntesis de Proteínas/genética , Estabilidad del ARN , ARN Mensajero/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Poliadenilación , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Poliadenilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Longer poly(A) tails improve translation in early development, but not in mature cells that have higher levels of the protein PABPC.
Asunto(s)
Oocitos , ARN MensajeroRESUMEN
Electrophoretic mobility shift assays (EMSAs) are among the most frequently used and straightforward experiments for studying protein-nucleic acid interactions. EMSAs rely on the principle that protein-nucleic acid complexes have reduced electrophoretic mobility in a native gel matrix compared to free nucleic acid due to their larger size and reduced negative charge. Therefore, bands for the protein-nucleic acid complexes are shifted in a gel and can be distinguished from free nucleic acids. EMSAs remain a popular technique since they do not require specialist equipment and the complexes formed are easily visualized. Furthermore, the technique can be adapted to enable various aspects of protein-nucleic acid interactions to be investigated, including sequence specificity, estimated binding affinity, and binding stoichiometry.
Asunto(s)
Ensayo de Cambio de Movilidad Electroforética/métodos , Ácidos Nucleicos/análisis , Proteínas/análisis , Resinas Acrílicas , Fenómenos Biofísicos , ADN/análisis , ADN/metabolismo , Ácidos Nucleicos/metabolismo , Unión Proteica , Proteínas/metabolismo , ARN/análisis , ARN/metabolismoRESUMEN
Cryo-electron microscopy of protein complexes often leads to moderate resolution maps (4-8â Å), with visible secondary-structure elements but poorly resolved loops, making model building challenging. In the absence of high-resolution structures of homologues, only coarse-grained structural features are typically inferred from these maps, and it is often impossible to assign specific regions of density to individual protein subunits. This paper describes a new method for overcoming these difficulties that integrates predicted residue distance distributions from a deep-learned convolutional neural network, computational protein folding using Rosetta, and automated EM-map-guided complex assembly. We apply this method to a 4.6â Å resolution cryoEM map of Fanconi Anemia core complex (FAcc), an E3 ubiquitin ligase required for DNA interstrand crosslink repair, which was previously challenging to interpret as it comprises 6557 residues, only 1897 of which are covered by homology models. In the published model built from this map, only 387 residues could be assigned to the specific subunits with confidence. By building and placing into density 42 deep-learning-guided models containing 4795 residues not included in the previously published structure, we are able to determine an almost-complete atomic model of FAcc, in which 5182 of the 6557 residues were placed. The resulting model is consistent with previously published biochemical data, and facilitates interpretation of disease-related mutational data. We anticipate that our approach will be broadly useful for cryoEM structure determination of large complexes containing many subunits for which there are no homologues of known structure.
RESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.