RESUMEN
LINE-1 retrotransposition is tightly restricted by layers of regulatory control, with epigenetic pathways being the best characterized. Looking at post-transcriptional regulation, we now show that LINE-1 mRNA 3' ends are pervasively uridylated in various human cellular models and in mouse testes. TUT4 and TUT7 uridyltransferases catalyze the modification and function in cooperation with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of the L1-ORF1p retrotransposon protein. Uridylation potently restricts LINE-1 retrotransposition by a multilayer mechanism depending on differential subcellular localization of the uridyltransferases. We propose that uridine residues added by TUT7 in the cytoplasm inhibit initiation of reverse transcription of LINE-1 mRNAs once they are reimported to the nucleus, whereas uridylation by TUT4, which is enriched in cytoplasmic foci, destabilizes mRNAs. These results provide a model for the post-transcriptional restriction of LINE-1, revealing a key physiological role for TUT4/7-mediated uridylation in maintaining genome stability.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , ARN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Uridina/metabolismo , Animales , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Ratones , Proteínas Nucleares/genética , Unión Proteica , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/genética , ARN Helicasas/metabolismo , Interferencia de ARN , ARN Nucleotidiltransferasas/antagonistas & inhibidores , ARN Nucleotidiltransferasas/genética , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Retroelementos/genéticaRESUMEN
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
Complete cytoplasmic polyadenosine tail (polyA-tail) deadenylation is thought to be essential for initiating mRNA decapping and subsequent degradation. To investigate this prevalent model, we conducted direct RNA sequencing of S. cerevisiae mRNAs derived from chase experiments under steady-state and stress condition. Subsequently, we developed a numerical model based on a modified gamma distribution function, which estimated the transcriptomic deadenylation rate at 10 A/min. A simplified independent method, based on the delineation of quantile polyA-tail values, showed a correlation between the decay and deadenylation rates of individual mRNAs, which appeared consistent within functional transcript groups and associated with codon optimality. Notably, these rates varied during the stress response. Detailed analysis of ribosomal protein-coding mRNAs (RPG mRNAs), constituting 40% of the transcriptome, singled out this transcript group. While deadenylation and decay of RPG mRNAs accelerated under heat stress, their degradation could proceed even when deadenylation was blocked, depending entirely on ongoing nuclear export. Our findings support the general primary function of deadenylation in dictating the onset of decapping, while also demonstrating complex relations between these processes.
RESUMEN
Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain that is common for many antiviral proteins. AbiA forms a dimer that distinguishes it from AbiK and Abi-P2, which form trimers/hexamers. We show the DNA polymerase activity of AbiA in an in vitro assay and demonstrate that it requires the presence of the HEPN domain which is enzymatically inactive. We validate our biochemical and structural results in vivo through bacteriophage infection assays. Finally, our in vivo results suggest that AbiA-mediated phage defense may not rely on AbiA-mediated cell death.
Asunto(s)
Bacteriófagos , Lactococcus , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacteriófagos/genética , Cristalografía por Rayos X , Lactococcus/virología , Lactococcus/genética , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , ADN Polimerasa Dirigida por ARN/metabolismo , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/genética , Relación Estructura-ActividadRESUMEN
Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures1,2. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.
Asunto(s)
Herpesvirus Humano 1/inmunología , ARN Bicatenario/inmunología , ARN Mitocondrial/inmunología , Animales , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/metabolismo , Exorribonucleasas/deficiencia , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Regulación de la Expresión Génica/inmunología , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/inmunología , Helicasa Inducida por Interferón IFIH1/metabolismo , Ratones , Ratones Endogámicos C57BL , Complejos Multienzimáticos/metabolismo , Mutación , Polirribonucleótido Nucleotidiltransferasa/metabolismo , ARN Helicasas/metabolismo , Análisis de la Célula Individual , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Synapses are the regions of the neuron that enable the transmission and propagation of action potentials on the cost of high energy consumption and elevated demand for mitochondrial ATP production. The rapid changes in local energetic requirements at dendritic spines imply the role of mitochondria in the maintenance of their homeostasis. Using global proteomic analysis supported with complementary experimental approaches, we show that an essential pool of mitochondrial proteins is locally produced at the synapse indicating that mitochondrial protein biogenesis takes place locally to maintain functional mitochondria in axons and dendrites. Furthermore, we show that stimulation of synaptoneurosomes induces the local synthesis of mitochondrial proteins that are transported to the mitochondria and incorporated into the protein supercomplexes of the respiratory chain. Importantly, in a mouse model of fragile X syndrome, Fmr1 KO mice, a common disease associated with dysregulation of synaptic protein synthesis, we observed altered morphology and respiration rates of synaptic mitochondria. That indicates that the local production of mitochondrial proteins plays an essential role in synaptic functions.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Animales , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Proteómica , SinapsisRESUMEN
MicroRNAs (miRNAs) control gene expression by regulating mRNA translation and stability. The CCR4-NOT complex is a key effector of miRNA function acting downstream of GW182/TNRC6 proteins. We show that miRNA-mediated repression requires the central region of CNOT1, the scaffold protein of CCR4-NOT. A CNOT1 domain interacts with CNOT9, which in turn interacts with the silencing domain of TNRC6 in a tryptophan motif-dependent manner. These interactions are direct, as shown by the structure of a CNOT9-CNOT1 complex with bound tryptophan. Another domain of CNOT1 with an MIF4G fold recruits the DEAD-box ATPase DDX6, a known translational inhibitor. Structural and biochemical approaches revealed that CNOT1 modulates the conformation of DDX6 and stimulates ATPase activity. Structure-based mutations showed that the CNOT1 MIF4G-DDX6 interaction is important for miRNA-mediated repression. These findings provide insights into the repressive steps downstream of the GW182/TNRC6 proteins and the role of the CCR4-NOT complex in posttranscriptional regulation in general.
Asunto(s)
ARN Helicasas DEAD-box/química , MicroARNs/genética , Proteínas Proto-Oncogénicas/química , Interferencia de ARN , Factores de Transcripción/química , Sustitución de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Complejos Multiproteicos/química , Mutagénesis Sitio-Dirigida , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3'-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3' CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH2 of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.
Asunto(s)
Proteínas Bacterianas/genética , Citosina/metabolismo , ARN Nucleotidiltransferasas/genética , ARN/genética , Aspergillus nidulans/genética , Proteínas Bacterianas/química , Sitios de Unión/genética , Cristalografía por Rayos X , Citosina/química , Modelos Moleculares , Poli A/química , Poli A/genética , ARN Nucleotidiltransferasas/química , Especificidad por SustratoRESUMEN
Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5' or 3' terminus of the molecule can serve as probes in hybridization-based analyses of in vivo degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases. In some applications, the placement of the label is not a concern, while in other cases it is required that the radioactive mark is located at the 5'- or 3'-end of the molecule. An unsurpassed method for 5'-end RNA labelling employs T4 polynucleotide kinase (PNK) and [γ-32P]ATP. In the case of 3'-end labelling, several different possibilities exist. However, they require the use of costly radionucleotide analogues. Previously, we characterized an untypical nucleotidyltransferase named CutA, which preferentially incorporates cytidines at the 3'-end of RNA substrates. Here, we demonstrate that this unusual feature can be used for the development of a novel, efficient, reproducible and economical method of RNA 3'-end labelling by CutA-mediated cytidine tailing. The labelling efficiency is comparable to that achieved with the most common method applied to date, i.e. [5'-32P]pCp ligation to the RNA 3'-terminus catalysed by T4 RNA ligase I. We show the utility of RNA substrates labelled using our new method in exemplary biochemical assays assessing directionality of two well-known eukaryotic exoribonucleases, namely Dis3 and Xrn1.
Asunto(s)
Nucleotidiltransferasas/química , ARN/química , Coloración y Etiquetado/métodos , Citidina Trifosfato/química , Técnicas In Vitro , Marcaje Isotópico/métodos , Nucleótidos/química , Radioisótopos de Fósforo , ARN/genética , ARN Ligasa (ATP)/química , Coloración y Etiquetado/normas , Especificidad por Sustrato , Uridina Trifosfato/químicaRESUMEN
Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and sorting include the TIM23 translocase responsible for the transfer of precursor proteins into the matrix, and the mitochondrial intermembrane space import and assembly (MIA) machinery required for the biogenesis of intermembrane space proteins. Dysfunction of mitochondrial protein sorting pathways results in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death. The cellular responses caused by accumulation of mitochondrial precursor proteins in the cytosol are mainly unknown. Here we present a comprehensive picture of the changes in the cellular transcriptome and proteome in response to a mitochondrial import defect and precursor over-accumulation stress. Pathways were identified that protect the cell against mitochondrial biogenesis defects by inhibiting protein synthesis and by activation of the proteasome, a major machine for cellular protein clearance. Proteasomal activity is modulated in proportion to the quantity of mislocalized mitochondrial precursor proteins in the cytosol. We propose that this type of unfolded protein response activated by mistargeting of proteins (UPRam) is beneficial for the cells. UPRam provides a means for buffering the consequences of physiological slowdown in mitochondrial protein import and for counteracting pathologies that are caused or contributed by mitochondrial dysfunction.
Asunto(s)
Citosol/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Mitocondrias/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Biosíntesis de Proteínas , Transporte de Proteínas/genética , Proteoma/genética , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico/genética , Transcriptoma , Respuesta de Proteína Desplegada/genéticaRESUMEN
Maintenance of mitochondrial gene expression is crucial for cellular homeostasis. Stress conditions may lead to a temporary reduction of mitochondrial genome copy number, raising the risk of insufficient expression of mitochondrial encoded genes. Little is known how compensatory mechanisms operate to maintain proper mitochondrial transcripts levels upon disturbed transcription and which proteins are involved in them. Here we performed a quantitative proteomic screen to search for proteins that sustain expression of mtDNA under stress conditions. Analysis of stress-induced changes of the human mitochondrial proteome led to the identification of several proteins with poorly defined functions among which we focused on C6orf203, which we named MTRES1 (Mitochondrial Transcription Rescue Factor 1). We found that the level of MTRES1 is elevated in cells under stress and we show that this upregulation of MTRES1 prevents mitochondrial transcript loss under perturbed mitochondrial gene expression. This protective effect depends on the RNA binding activity of MTRES1. Functional analysis revealed that MTRES1 associates with mitochondrial RNA polymerase POLRMT and acts by increasing mitochondrial transcription, without changing the stability of mitochondrial RNAs. We propose that MTRES1 is an example of a protein that protects the cell from mitochondrial RNA loss during stress.
Asunto(s)
Perfilación de la Expresión Génica , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteómica/métodos , Proteínas de Unión al ARN/metabolismo , Transcripción Genética/genética , Secuencia de Aminoácidos , Genes Mitocondriales/genética , Células HEK293 , Células HeLa , Humanos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteoma/genética , Proteoma/metabolismo , ARN Mitocondrial/genética , Proteínas de Unión al ARN/genética , Homología de Secuencia de Aminoácido , Estrés FisiológicoRESUMEN
The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.
Asunto(s)
Mycobacterium tuberculosis/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , ARN/análisis , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complejos Multienzimáticos , Mycobacterium smegmatis/metabolismo , Polirribonucleótido Nucleotidiltransferasa/genética , Proteoma , Proteómica , ARN/química , ARN Helicasas/metabolismo , Estabilidad del ARN , ARN Bacteriano/metabolismo , Ribonucleasa III/metabolismo , Ribonucleasas/metabolismo , Tiouridina/química , TranscriptomaRESUMEN
Pre-rRNA processing generates mature 18S, 5.8S, and 28S/25S rRNAs through multistage removal of surrounding 5'-ETS/3'-ETS and intervening ITS1/ITS2 segments. Endonucleolytic activities release by-products, which need to be eliminated. Here, we investigated the interplay of exosome-associated 3'-5' exonucleases DIS3 and RRP6 in rRNA processing and by-product elimination in human cells. In agreement with previous reports, we observed accumulation of 5.8S and 18S precursors upon dysfunction of these enzymes. However, none of these phenotypes was so pronounced as previously overlooked accumulation of short RNA species derived from 5'-ETS (01/A'-A0), in cells with nonfunctional DIS3. We demonstrate that removal of 01/A'-A0 is independent of the XRN2 5'-3' exonucleolytic activity. Instead, it proceeds rapidly after A0 cleavage and occurs exclusively in the 3'-5' direction in several phases-following initiation by an unknown nuclease, the decay is executed by RRP6 with some contribution of DIS3, whereas the ultimate phase involves predominantly DIS3. Our data shed new light onto the role of human exosome in 5'-ETS removal. Furthermore, although 01/A'-A0 degradation involves the action of two nucleases associated with the exosome ring, similarly to 5.8S 3'-end maturation, it is likely that contrary to the latter process, RRP6 acts prior to or redundantly with DIS3.
Asunto(s)
Exorribonucleasas/química , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Precursores del ARN/química , Procesamiento Postranscripcional del ARN/genética , Núcleo Celular/química , Núcleo Celular/genética , Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Exosomas/química , Exosomas/enzimología , Humanos , Precursores del ARN/genética , Ribonucleasas/química , Ribonucleasas/genéticaRESUMEN
Plasmids are mobile genetics elements that play an important role in the environmental adaptation of microorganisms. Although plasmids are usually analyzed in cultured microorganisms, there is a need for methods that allow for the analysis of pools of plasmids (plasmidomes) in environmental samples. To that end, several molecular biology and bioinformatics methods have been developed; however, they are limited to environments with low diversity and cannot recover large plasmids. Here, we present PlasFlow, a novel tool based on genomic signatures that employs a neural network approach for identification of bacterial plasmid sequences in environmental samples. PlasFlow can recover plasmid sequences from assembled metagenomes without any prior knowledge of the taxonomical or functional composition of samples with an accuracy up to 96%. It can also recover sequences of both circular and linear plasmids and can perform initial taxonomical classification of sequences. Compared to other currently available tools, PlasFlow demonstrated significantly better performance on test datasets. Analysis of two samples from heavy metal-contaminated microbial mats revealed that plasmids may constitute an important fraction of their metagenomes and carry genes involved in heavy-metal homeostasis, proving the pivotal role of plasmids in microorganism adaptation to environmental conditions.
Asunto(s)
Bacterias/genética , Biología Computacional/métodos , Metagenoma/genética , Metagenómica/métodos , Plásmidos/genética , Algoritmos , Bacterias/clasificación , Genómica/métodos , Filogenia , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/métodosRESUMEN
C16orf57 encodes a human protein of unknown function, and mutations in the gene occur in poikiloderma with neutropenia (PN), which is a rare, autosomal recessive disease. Interestingly, mutations in C16orf57 were also observed among patients diagnosed with Rothmund-Thomson syndrome (RTS) and dyskeratosis congenita (DC), which are caused by mutations in genes involved in DNA repair and telomere maintenance. A genetic screen in Saccharomyces cerevisiae revealed that the yeast ortholog of C16orf57, USB1 (YLR132C), is essential for U6 small nuclear RNA (snRNA) biogenesis and cell viability. Usb1 depletion destabilized U6 snRNA, leading to splicing defects and cell growth defects, which was suppressed by the presence of multiple copies of the U6 snRNA gene SNR6. Moreover, Usb1 is essential for the generation of a unique feature of U6 snRNA; namely, the 3'-terminal phosphate. RNAi experiments in human cells followed by biochemical and functional analyses confirmed that, similar to yeast, C16orf57 encodes a protein involved in the 2',3'-cyclic phosphate formation at the 3' end of U6 snRNA. Advanced bioinformatics predicted that C16orf57 encodes a phosphodiesterase whose putative catalytic activity is essential for its function in vivo. Our results predict an unexpected molecular basis for PN, DC, and RTS and provide insight into U6 snRNA 3' end formation.
Asunto(s)
Mutación , Neutropenia/genética , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Procesamiento de Término de ARN 3'/genética , ARN Nuclear Pequeño/metabolismo , Síndrome Rothmund-Thomson/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Neutropenia/enzimología , Hidrolasas Diéster Fosfóricas/química , Estructura Terciaria de Proteína , Interferencia de ARN , Estabilidad del ARN/genética , Síndrome Rothmund-Thomson/enzimología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Noncanonical RNA nucleotidyltransferases (NTases), including poly(A), poly(U) polymerases (PAPs/PUPs), and C/U-adding enzymes, modify 3'-ends of different transcripts affecting their functionality and stability. They contain PAP/OAS1 substrate-binding domain (SBD) with inserted NTase domain. Aspergillus nidulans CutA (AnCutA), synthesizes C/U-rich 3'-terminal extensions in vivo. Here, using high-throughput sequencing of the 3'-RACE products for tails generated by CutA proteins in vitro in the presence of all four NTPs, we show that even upon physiological ATP excess synthesized tails indeed contain an unprecedented number of cytidines interrupted by uridines and stretches of adenosines, and that the majority end with two cytidines. Strikingly, processivity assays documented that in the presence of CTP as a sole nucleotide, the enzyme terminates after adding two cytidines only. Comparison of our CutA 3D model to selected noncanonical NTases of known structures revealed substantial differences in the nucleotide recognition motif (NRM) within PAP/OAS1 SBD. We demonstrate that CutA specificity toward CTP can be partially changed to PAP or PUP by rational mutagenesis within NRM and, analogously, Cid1 PUP can be converted into a C/U-adding enzyme. Collectively, we suggest that a short cluster of amino acids within NRM is a determinant of NTases' substrate preference, which may allow us to predict their specificity.
Asunto(s)
Aspergillus nidulans/enzimología , Biología Computacional/métodos , Citidina Trifosfato/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Secuencia de Aminoácidos , Citidina/química , Citidina Trifosfato/química , Modelos Moleculares , Homología de Secuencia , Especificidad por SustratoRESUMEN
The RNA exosome is a conserved degradation machinery, which obtains full activity only when associated with cofactors. The most prominent activator of the yeast nuclear exosome is the RNA helicase Mtr4p, acting in the context of the Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex. The existence of a similar activator(s) in humans remains elusive. By establishing an interaction network of the human nuclear exosome, we identify the trimeric Nuclear Exosome Targeting (NEXT) complex, containing hMTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7. ZCCHC8 and RBM7 are excluded from nucleoli, and consistently NEXT is specifically required for the exosomal degradation of promoter upstream transcripts (PROMPTs). We also detect putative homolog TRAMP subunits hTRF4-2 (Trf4p) and ZCCHC7 (Air2p) in hRRP6 and hMTR4 precipitates. However, at least ZCCHC7 function is restricted to nucleoli. Our results suggest that human nuclear exosome degradation pathways comprise modules of spatially organized cofactors that diverge from the yeast model.
Asunto(s)
Proteínas Portadoras/fisiología , Modelos Biológicos , Proteínas Nucleares/fisiología , ARN Helicasas/fisiología , Proteínas de Unión al ARN/fisiología , Ribonucleasas/metabolismo , Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Nucléolo Celular/enzimología , Nucléolo Celular/metabolismo , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/metabolismo , ADN Polimerasa Dirigida por ADN/análisis , ADN Polimerasa Dirigida por ADN/metabolismo , Exorribonucleasas/análisis , Exorribonucleasas/metabolismo , Exorribonucleasas/fisiología , Complejo Multienzimático de Ribonucleasas del Exosoma , Humanos , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , ARN Helicasas/análisis , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/análisis , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Ichthyosis and neurological involvement occur in relatively few known Mendelian disorders caused by mutations in genes relevant both for epidermis and neural function. OBJECTIVES: To identify the cause of a similar phenotype of ichthyotic keratoderma, spasticity, mild hypomyelination (on MRI) and dysmorphic features (IKSHD) observed in two unrelated paediatric probands without family history of disease. METHODS: Whole exome sequencing was performed in both patients. The functional effect of prioritised variant in ELOVL1 (very-long-chain fatty acids (VLCFAs) elongase) was analysed by VLCFA profiling by gas chromatography-mass spectrometry in stably transfected HEK2932 cells and in cultured patient's fibroblasts. RESULTS: Probands shared novel heterozygous ELOVL1 p.Ser165Phe mutation (de novo in one family, while in the other family, father could not be tested). In transfected cells p.Ser165Phe: (1) reduced levels of FAs C24:0-C28:0 and C26:1 with the most pronounced effect for C26:0 (P=7.8×10-6 vs HEK293 cells with wild type (wt) construct, no difference vs naïve HEK293) and (2) increased levels of C20:0 and C22:0 (P=6.3×10-7, P=1.2×10-5, for C20:0 and C22:0, respectively, comparison vs HEK293 cells with wt construct; P=2.2×10-7, P=1.9×10-4, respectively, comparison vs naïve HEK293). In skin fibroblasts, there was decrease of C26:1 (P=0.014), C28:0 (P=0.001) and increase of C20:0 (P=0.033) in the patient versus controls. There was a strong correlation (r=0.92, P=0.008) between the FAs profile of patient's fibroblasts and that of p.Ser165Phe transfected HEK293 cells. Serum levels of C20:0-C26:0 FAs were normal, but the C24:0/C22:0 ratio was decreased. CONCLUSION: The ELOVL1 p.Ser165Phe mutation is a likely cause of IKSHD.
Asunto(s)
Acetiltransferasas/genética , Trastorno Dismórfico Corporal/genética , Ictiosis/genética , Enfermedades del Sistema Nervioso/genética , Adolescente , Trastorno Dismórfico Corporal/complicaciones , Trastorno Dismórfico Corporal/diagnóstico por imagen , Trastorno Dismórfico Corporal/fisiopatología , Niño , Preescolar , Elongasas de Ácidos Grasos , Células HEK293 , Humanos , Ictiosis/complicaciones , Ictiosis/diagnóstico por imagen , Ictiosis/fisiopatología , Lactante , Imagen por Resonancia Magnética , Masculino , Mutación , Enfermedades del Sistema Nervioso/complicaciones , Enfermedades del Sistema Nervioso/diagnóstico por imagen , Enfermedades del Sistema Nervioso/fisiopatología , Secuenciación del ExomaRESUMEN
The exosome complex is a major eukaryotic exoribonuclease that requires the SKI complex for its activity in the cytoplasm. In yeast, the Ski7 protein links both complexes, whereas a functional equivalent of the Ski7 has remained unknown in the human genome. Proteomic analysis revealed that a previously uncharacterized short splicing isoform of HBS1L (HBS1LV3) is the long-sought factor linking the exosome and SKI complexes in humans. In contrast, the canonical HBS1L variant, HBS1LV1, which acts as a ribosome dissociation factor, does not associate with the exosome and instead interacts with the mRNA surveillance factor PELOTA. Interestingly, both HBS1LV1 and HBS1LV3 interact with the SKI complex and HBS1LV1 seems to antagonize SKI/exosome supercomplex formation. HBS1LV3 contains a unique C-terminal region of unknown structure, with a conserved RxxxFxxxL motif responsible for exosome binding and may interact with the exosome core subunit RRP43 in a way that resembles the association between Rrp6 RNase and Rrp43 in yeast. HBS1LV3 or the SKI complex helicase (SKI2W) depletion similarly affected the transcriptome, deregulating multiple genes. Furthermore, half-lives of representative upregulated mRNAs were increased, supporting the involvement of HBS1LV3 and SKI2W in the same mRNA degradation pathway, essential for transcriptome homeostasis in the cytoplasm.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Proteínas de Unión al GTP/metabolismo , Sitios de Unión , Citoplasma/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Proteínas de Unión al GTP/genética , Células HEK293 , Humanos , Modelos Moleculares , Conformación Proteica en Hélice alfa , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Empalme del ARN , Estabilidad del ARN , ARN Mensajero/metabolismoRESUMEN
Human DIS3, the nuclear catalytic subunit of the exosome complex, contains exonucleolytic and endonucleolytic active domains. To identify DIS3 targets genome-wide, we combined comprehensive transcriptomic analyses of engineered HEK293 cells that expressed mutant DIS3, with Photoactivatable Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) experiments. In cells expressing DIS3 with both catalytic sites mutated, RNAs originating from unannotated genomic regions increased â¼2.5-fold, covering â¼70% of the genome and allowing for thousands of novel transcripts to be discovered. Previously described pervasive transcription products, such as Promoter Upstream Transcripts (PROMPTs), accumulated robustly upon DIS3 dysfunction, representing a significant fraction of PAR-CLIP reads. We have also detected relatively long putative premature RNA polymerase II termination products of protein-coding genes whose levels in DIS3 mutant cells can exceed the mature mRNAs, indicating that production of such truncated RNA is a common phenomenon. In addition, we found DIS3 to be involved in controlling the formation of paraspeckles, nuclear bodies that are organized around NEAT1 lncRNA, whose short form was overexpressed in cells with mutated DIS3. Moreover, the DIS3 mutations resulted in misregulation of expression of â¼50% of transcribed protein-coding genes, probably as a secondary effect of accumulation of various noncoding RNA species. Finally, cells expressing mutant DIS3 accumulated snoRNA precursors, which correlated with a strong PAR-CLIP signal, indicating that DIS3 is the main snoRNA-processing enzyme. EXOSC10 (RRP6) instead controls the levels of the mature snoRNAs. Overall, we show that DIS3 has a major nucleoplasmic function in shaping the human RNA polymerase II transcriptome.