RESUMO
CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.
Assuntos
Doenças do Sistema Nervoso Central/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Fosfotransferases/metabolismo , RNA de Transferência/metabolismo , Fatores de Transcrição/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Animais , Doenças do Sistema Nervoso Central/patologia , Cérebro/patologia , Pré-Escolar , Endorribonucleases/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos CBA , Microcefalia/genética , Doenças do Sistema Nervoso Periférico/patologia , RNA de Transferência/genética , Proteínas de Ligação a RNARESUMO
The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , RNA Ligase (ATP)/metabolismo , RNA de Transferência/metabolismo , Animais , Antioxidantes/fisiologia , Domínio Catalítico , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD/metabolismo , NADP/metabolismo , Oxirredução , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/fisiologia , RNA Ligase (ATP)/química , RNA Ligase (ATP)/genética , Splicing de RNA/genética , Splicing de RNA/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.
Assuntos
Neurônios Motores/metabolismo , Neurônios Motores/patologia , RNA de Transferência de Tirosina/metabolismo , Fatores de Transcrição/metabolismo , Esclerose Lateral Amiotrófica , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/patologia , Morte Celular , Diafragma/inervação , Perda do Embrião , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Éxons/genética , Feminino , Fibroblastos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Atrofia Muscular Espinal , Doenças Neuromusculares/metabolismo , Doenças Neuromusculares/patologia , Estresse Oxidativo , Processamento Pós-Transcricional do RNA , RNA de Transferência de Tirosina/genética , Proteínas de Ligação a RNA , Respiração , Nervos Espinhais/citologia , Fatores de Transcrição/deficiência , Proteína Supressora de Tumor p53/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMO
The process of tRNA splicing entails removal of an intron by TSEN (tRNA-splicing endonuclease) and ligation of the resulting exon halves to generate functional tRNAs. In mammalian cells, the RNA kinase CLP1 (cleavage and polyadenylation factor I subunit) associates with TSEN and phosphorylates the 3' exon at the 5' end in vitro, suggesting a role for CLP1 in tRNA splicing. Interestingly, recent data suggest that the ATP-binding and/or hydrolysis capacity of CLP1 is required to enhance pre-tRNA cleavage. In vivo, the lack of CLP1 kinase activity leads to progressive motor neuron loss and accumulation of novel 5' leader-5' exon tRNA fragments. We have extended the investigation of the biochemical requirements in pre-tRNA splicing and found that ß-γ-hydrolysable ATP is crucial for the productive generation of exon halves. In addition, we provide evidence that phosphorylation of the TSEN complex components supports efficient pre-tRNA cleavage. Taken together, our data improve the mechanistic understanding of mammalian pre-tRNA processing and its regulation.
Assuntos
Trifosfato de Adenosina/metabolismo , Endorribonucleases/metabolismo , Íntrons , Precursores de RNA/genética , Splicing de RNA , RNA de Transferência/genética , Animais , Humanos , Hidrólise , Camundongos , FosforilaçãoRESUMO
RNA interference allows the analysis of gene function by introducing synthetic, short interfering RNAs (siRNAs) into cells. In contrast to siRNA and microRNA duplexes generated endogenously by the RNaseIII endonuclease Dicer, synthetic siRNAs display a 5' OH group. However, to become incorporated into the RNA-induced silencing complex (RISC) and mediate target RNA cleavage, the guide strand of an siRNA needs to display a phosphate group at the 5' end. The identity of the responsible kinase has so far remained elusive. Monitoring siRNA phosphorylation, we applied a chromatographic approach that resulted in the identification of the protein hClp1 (human Clp1), a known component of both transfer RNA splicing and messenger RNA 3'-end formation machineries. Here we report that the kinase hClp1 phosphorylates and licenses synthetic siRNAs to become assembled into RISC for subsequent target RNA cleavage. More importantly, we reveal the physiological role of hClp1 as the RNA kinase that phosphorylates the 5' end of the 3' exon during human tRNA splicing, allowing the subsequent ligation of both exon halves by an unknown tRNA ligase. The investigation of this novel enzymatic activity of hClp1 in the context of mRNA 3'-end formation, where no RNA phosphorylation event has hitherto been predicted, remains a challenge for the future.
Assuntos
Éxons/genética , Fosfotransferases/metabolismo , RNA Interferente Pequeno/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Fatores de Transcrição/metabolismo , Inativação Gênica , Humanos , Proteínas Nucleares , Fosforilação , Fosfotransferases/deficiência , Fosfotransferases/genética , Fosfotransferases/imunologia , Interferência de RNA , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Complexo de Inativação Induzido por RNA/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of functional redundancies can create de novo vulnerabilities that are specific to cancer cells. Here, we systematically search for cancer-relevant paralog interactions using CRISPR screens and publicly available loss-of-function datasets. Our analysis reveals >2,000 candidate dependencies, several of which we validate experimentally, including CSTF2-CSTF2T, DNAJC15-DNAJC19, FAM50A-FAM50B, and RPP25-RPP25L. We provide evidence that RPP25L can physically and functionally compensate for the absence of RPP25 as a member of the RNase P/MRP complexes in tRNA processing. Our analysis also reveals unexpected redundancies between sex chromosome genes. We show that chrX- and chrY-encoded paralogs, such as ZFX-ZFY, DDX3X-DDX3Y, and EIF1AX-EIF1AY, are functionally linked. Tumor cell lines from male patients with loss of chromosome Y become dependent on the chrX-encoded gene. We propose targeting of chrX-encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y chromosome.
Assuntos
Neoplasias , Oncogenes , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Antígenos de Histocompatibilidade Menor/metabolismo , Neoplasias/genética , Proteínas de Ligação a RNA/genética , Cromossomos Sexuais/metabolismo , Cromossomo X , Cromossomo YRESUMO
RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B, and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair, and mRNA transport. Here, we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex and provide a structural framework for understanding its functions in cellular RNA metabolism.
Assuntos
Proteínas/química , RNA Ligase (ATP)/química , Transativadores/química , Humanos , Estrutura MolecularRESUMO
Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.
Assuntos
Doenças Cerebelares/metabolismo , Endonucleases/metabolismo , Mutação , Precursores de RNA/metabolismo , Splicing de RNA , RNA de Transferência/metabolismo , Animais , Doenças Cerebelares/genética , Cristalografia por Raios X , Endonucleases/química , Endonucleases/genética , Endorribonucleases/química , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células HEK293 , Humanos , Íntrons/genética , Conformação Proteica , Multimerização Proteica , Precursores de RNA/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Células Sf9 , SpodopteraRESUMO
Recognition of foreign DNA by cytosolic innate immune receptors triggers the production of IFN-beta. However, it is unclear whether different types of DNA ligands are recognized by similar receptors and whether the resulting response is distinct from the endosomal TLR response. To address these questions, we compared the two most commonly used types of DNA ligands (IFN-stimulatory DNA (ISD) and poly(dAdT)) and assessed the minimal structural requirements for stimulatory capacity in RAW264.7 cells. Gene expression signatures and competition experiments suggest that ISD and poly(dAdT) are qualitatively indistinguishable and differ from the CpG-containing oligonucleotides triggering the TLR9 pathway. Structure - activity relationship analyses revealed that a minimal length of two helical turns is sufficient for ISD-mediated IFN-beta induction, while phosphorylation at the 5'-end is dispensable. Altogether, our data suggest that, in murine macrophages, only one major cytosolic DNA recognition pathway is operational.
Assuntos
DNA/genética , Interferon beta/genética , Macrófagos/metabolismo , Transdução de Sinais , Receptor Toll-Like 9/metabolismo , Animais , Linhagem Celular , Quimiocina CXCL11/genética , Ilhas de CpG/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/efeitos dos fármacos , Immunoblotting , Interferon beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/genética , Fosforilação , Poli dA-dT/genética , Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serpinas/genética , TransfecçãoRESUMO
RNA molecules are frequently modified with a terminal 2',3'-cyclic phosphate group as a result of endonuclease cleavage, exonuclease trimming, or de novo synthesis. During pre-transfer RNA (tRNA) and unconventional messenger RNA (mRNA) splicing, 2',3'-cyclic phosphates are substrates of the tRNA ligase complex, and their removal is critical for recycling of tRNAs upon ribosome stalling. We identified the predicted deadenylase angel homolog 2 (ANGEL2) as a human phosphatase that converts 2',3'-cyclic phosphates into 2',3'-OH nucleotides. We analyzed ANGEL2's substrate preference, structure, and reaction mechanism. Perturbing ANGEL2 expression affected the efficiency of pre-tRNA processing, X-box-binding protein 1 (XBP1) mRNA splicing during the unfolded protein response, and tRNA nucleotidyltransferase 1 (TRNT1)-mediated CCA addition onto tRNAs. Our results indicate that ANGEL2 is involved in RNA pathways that rely on the ligation or hydrolysis of 2',3'-cyclic phosphates.
Assuntos
Exorribonucleases/química , Nucleotidases/química , Ribonucleases/química , Cristalografia por Raios X , Exorribonucleases/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Nucleotidases/genética , Estrutura Secundária de Proteína , Precursores de RNA , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/genética , Especificidade por Substrato , Proteína 1 de Ligação a X-Box/genéticaRESUMO
During mitosis, in most eukaryotes, cohesin is removed from chromosomes in two steps. A paper in the March issue of Molecular Cell identifies Polo-like kinase as a key regulator for the first step that releases much of cohesin during prophase.
Assuntos
Segregação de Cromossomos/fisiologia , Proteínas de Drosophila , Prófase/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Xenopus , Animais , Proteínas de Ciclo CelularRESUMO
Polymerase δ is essential for eukaryotic genome duplication and synthesizes DNA at both the leading and lagging strands. The polymerase δ complex is a heterotetramer comprising the catalytic subunit POLD1 and the accessory subunits POLD2, POLD3, and POLD4. Beyond DNA replication, the polymerase δ complex has emerged as a central element in genome maintenance. The essentiality of polymerase δ has constrained the generation of polymerase δ-knockout cell lines or model organisms and, therefore, the understanding of the complexity of its activity and the function of its accessory subunits. To our knowledge, no germline biallelic mutations affecting this complex have been reported in humans. In patients from 2 independent pedigrees, we have identified what we believe to be a novel syndrome with reduced functionality of the polymerase δ complex caused by germline biallelic mutations in POLD1 or POLD2 as the underlying etiology of a previously unknown autosomal-recessive syndrome that combines replicative stress, neurodevelopmental abnormalities, and immunodeficiency. Patients' cells showed impaired cell-cycle progression and replication-associated DNA lesions that were reversible upon overexpression of polymerase δ. The mutations affected the stability and interactions within the polymerase δ complex or its intrinsic polymerase activity. We believe our discovery of human polymerase δ deficiency identifies the central role of this complex in the prevention of replication-related DNA lesions, with particular relevance to adaptive immunity.
Assuntos
DNA Polimerase III/deficiência , DNA Polimerase III/genética , Mutação em Linhagem Germinativa , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Adolescente , Alelos , Substituição de Aminoácidos , DNA Polimerase III/química , Replicação do DNA/genética , Estabilidade Enzimática/genética , Genes Recessivos , Humanos , Masculino , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/deficiência , Complexos Multienzimáticos/genética , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento/enzimologia , Transtornos do Neurodesenvolvimento/genética , Linhagem , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Adulto JovemRESUMO
BACKGROUND: Cohesion between sister chromatids is promoted by the chromosomal cohesin complex that forms a proteinaceous ring, large enough in principle to embrace two sister strands. The mechanism by which cohesin binds to DNA, and how sister chromatid cohesion is established, is unknown. RESULTS: Biochemical studies of cohesin have largely been limited to protein isolated from soluble cellular fractions. Here, we characterize cohesin purified from budding yeast chromatin, suggesting that chromosomal cohesin is sufficiently described by its known distinctive ring structure. We present evidence that the two Smc subunits of cohesin by themselves form a ring, closed at interacting ATPase head domains. A motif in the Smc1 subunit implicated in ATP hydrolysis is essential for loading cohesin onto DNA. In addition to functional ATPase heads, an intact cohesin ring structure is indispensable for DNA binding, suggesting that ATP hydrolysis may be coupled to DNA transport into the cohesin ring. DNA is released in anaphase when separase cleaves cohesin's Scc1 subunit. We show that a cleavage fragment of Scc1 disrupts the interaction between the two Smc heads, thereby opening the ring. CONCLUSIONS: We present a model for cohesin binding to chromatin by ATP hydrolysis-dependent transport of DNA into the cohesin ring. After DNA replication, two DNA strands may be trapped to promote sister chromatid cohesion. In anaphase, Scc1 cleavage opens the ring to release sister chromatids.
Assuntos
Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Modelos Químicos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Cromatografia em Gel , Proteínas Cromossômicas não Histona , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas , Hidrólise , Proteínas Nucleares/isolamento & purificação , Fosfoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomycetales/genética , Saccharomycetales/metabolismo , Coloração pela Prata , CoesinasRESUMO
Defects in RNA metabolic pathways are well-established causes for neurodegenerative disorders. Several mutations in genes involved in pre-messenger RNA (pre-mRNA) and tRNA metabolism, RNA stability and protein translation have been linked to motor neuron diseases. Our study on a mouse carrying a catalytically inactive version of the RNA kinase CLP1, a component of the tRNA splicing endonuclease complex, revealed a neurological disorder characterized by progressive loss of lower spinal motor neurons. Surprisingly, mutant mice accumulate a novel class of tRNA-derived fragments. In addition, patients with homozygous missense mutations in CLP1 (R140H) were recently identified who suffer from severe motor-sensory defects, cortical dysgenesis and microcephaly, and exhibit alterations in transfer RNA (tRNA) splicing. Here, we review functions of CLP1 in different RNA pathways and provide hypotheses on the role of the tRNA splicing machinery in the generation of tRNA fragments and the molecular links to neurodegenerative disorders. We further immerse the biology of tRNA splicing into topics of (t)RNA metabolism and oxidative stress, putting forward the idea that defects in tRNA processing leading to tRNA fragment accumulation might trigger the development of neurodegenerative diseases.
Assuntos
Doenças Neurodegenerativas/fisiopatologia , Splicing de RNA , RNA de Transferência/metabolismo , Fatores de Transcrição/metabolismo , Animais , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estresse Oxidativo , Proteínas de Ligação a RNA , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Splicing of mammalian precursor transfer RNA (tRNA) molecules involves two enzymatic steps. First, intron removal by the tRNA splicing endonuclease generates separate 5' and 3' exons. In animals, the second step predominantly entails direct exon ligation by an elusive RNA ligase. Using activity-guided purification of tRNA ligase from HeLa cell extracts, we identified HSPC117, a member of the UPF0027 (RtcB) family, as the essential subunit of a tRNA ligase complex. RNA interference-mediated depletion of HSPC117 inhibited maturation of intron-containing pre-tRNA both in vitro and in living cells. The high sequence conservation of HSPC117/RtcB proteins is suggestive of RNA ligase roles of this protein family in various organisms.
Assuntos
Proteínas/química , Proteínas/metabolismo , RNA Ligase (ATP)/química , RNA Ligase (ATP)/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA de Transferência/metabolismo , Sequência de Aminoácidos , Éxons , Células HeLa , Humanos , Íntrons , Dados de Sequência Molecular , Proteínas/isolamento & purificação , Interferência de RNA , RNA Ligase (ATP)/isolamento & purificação , Spliceossomos/metabolismoRESUMO
The RNA-induced silencing complex (RISC) is the effector complex in the RNA interference (RNAi) pathway. In order to become assembled into RISC, synthetic small interfering RNAs (siRNAs) are phosphorylated at the 5' end upon transfection into cells. The enzymatic activity responsible for this phosphorylation event has so far remained elusive. Using a classical chromatographic approach, we recently identified and characterized hClp1 as the "siRNA-kinase" in HeLa cells. hClp1 is in fact a general RNA-kinase, and a component of the tRNA splicing endonuclease and the mRNA 3' end formation machinery. We discuss the relevance of this finding, and provide further views and perspectives for the analysis of hClp1 in tRNA splicing, mRNA cleavage and polyadenylation and other RNA metabolic processes in which hClp1 might play a role.
Assuntos
Proteínas Nucleares/metabolismo , Fosfotransferases/metabolismo , RNA/metabolismo , Fatores de Transcrição/metabolismo , Células HeLa , Humanos , Conformação de Ácido Nucleico , Processamento de Terminações 3' de RNA , Interferência de RNA , Splicing de RNA/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genéticaRESUMO
Cohesion between sister chromatids in eukaryotes is mediated by the evolutionarily conserved cohesin complex. Cohesin forms a proteinaceous ring, large enough to trap pairs of replicated sister chromatids. The circumference consists of the Smc1 and Smc3 subunits, while Scc1 is thought to close the ring by bridging the Smc (structural maintenance of chromosomes) ATPase head domains. Little is known about two additional subunits, Scc3 and Pds5, and about possible conformational changes of the complex during the cell cycle. We have employed fluorescence resonance energy transfer (FRET) to analyse interactions within the cohesin complex in live budding yeast. These experiments reveal an unexpected geometry of Scc1 at the Smc heads, and suggest that Pds5 plays a role at the Smc hinge on the opposite side of the ring. Key subunit interactions, including close proximity of the two ATPase heads, are constitutive throughout the cell cycle. This depicts cohesin as a stable molecular machine undergoing only transient conformational changes during binding and dissociation from chromosomes. Using FRET, we did not observe interactions between more than one cohesin complex in vivo.