RESUMEN
N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.
Asunto(s)
Acetilación , Citidina/análogos & derivados , Células Eucariotas/metabolismo , Evolución Molecular , ARN/química , ARN/metabolismo , Archaea/química , Archaea/citología , Archaea/genética , Archaea/crecimiento & desarrollo , Secuencia Conservada , Microscopía por Crioelectrón , Citidina/metabolismo , Células Eucariotas/citología , Células HeLa , Humanos , Modelos Moleculares , Acetiltransferasas N-Terminal/metabolismo , ARN de Archaea/química , ARN de Archaea/genética , Proteínas de Unión al ARN/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Ribosomas/ultraestructura , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , TemperaturaRESUMEN
Toll-like receptor 7 (TLR7) is a single-stranded RNA (ssRNA) sensor in innate immunity and also responds to guanosine and chemical ligands, such as imidazoquinoline compounds. However, TLR7 activation mechanism by these ligands remain largely unknown. Here, we generated crystal structures of three TLR7 complexes, and found that all formed an activated m-shaped dimer with two ligand-binding sites. The first site conserved in TLR7 and TLR8 was used for small ligand-binding essential for its activation. The second site spatially distinct from that of TLR8 was used for a ssRNA-binding that enhanced the affinity of the first-site ligands. The first site preferentially recognized guanosine and the second site specifically bound to uridine moieties in ssRNA. Our structural, biochemical, and mutagenesis studies indicated that TLR7 is a dual receptor for guanosine and uridine-containing ssRNA. Our findings have important implications for understanding of TLR7 function, as well as for therapeutic manipulation of TLR7 activation.
Asunto(s)
Guanosina/metabolismo , ARN/metabolismo , Receptor Toll-Like 7/química , Receptor Toll-Like 7/metabolismo , Animales , Sitios de Unión/inmunología , Línea Celular , Drosophila , Guanosina/inmunología , Células HEK293 , Humanos , Inmunidad Innata/inmunología , Ligandos , Macaca mulatta , ARN/inmunología , Receptor Toll-Like 7/inmunologíaRESUMEN
mRNA-based medicines are a promising modality for preventing virus-caused illnesses, including COVID-19, and treating various types of cancer and genetic diseases. To develop such medicines, methods to characterize long mRNA molecules are needed for quality control and metabolic analysis. Here, we developed an analytical platform based on isotope-dilution liquid chromatography-mass spectrometry (LC-MS) that quantitatively characterizes long, modified mRNAs by comparing them to a stable isotope-labeled reference with an identical sequence to that of the target medicine. This platform also includes database searching using the mass spectra as a query, which allowed us to confirm the primary structures of 200 to 4300 nt mRNAs including chemical modifications, with sequence coverage at 100%, to detect/identify defects in the sequences, and to define the efficiencies of the 5'-capping and integrity of the polyadenylated tail. Our findings indicated that this platform should be valuable for quantitatively characterizing mRNA vaccines and other mRNA medicines.
Asunto(s)
COVID-19 , Humanos , Indicadores y Reactivos , Espectrometría de Masas/métodos , Cromatografía Liquida/métodos , Estándares de Referencia , Isótopos , Marcaje Isotópico/métodosRESUMEN
Myokines, secreted factors from skeletal muscle, act locally on muscle cells or satellite cells, which is important in regulating muscle mass and function. Here, we found platelet-derived growth factor subunit B (PDGF-B) is constitutively secreted from muscle cells without muscle contraction. Furthermore, PDGF-B secretion increased with myoblast to myotube differentiation. To examine the role of PDGF-B as a paracrine or autocrine myokine, myoblasts or myotubes were treated with PDGF-B. As a result, myoblast proliferation was significantly enhanced via several signaling pathways. Intriguingly, myotubes treated with PDGF-B showed enhanced maturation as indicated by their increased myotube diameter, myosin heavy chain expression, and strengthened contractile force. These findings suggest that PDGF-B is constitutively secreted by myokines to enhance myoblast proliferation and myotube maturation, which may contribute to skeletal muscle regeneration.
Asunto(s)
Fibras Musculares Esqueléticas , Células Satélite del Músculo Esquelético , Diferenciación Celular/fisiología , Proliferación Celular , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético , Transducción de Señal , Animales , RatonesRESUMEN
Many ion channels and receptor proteins are potential targets for new drugs. However, standard methods for profiling these integral membrane proteins (IMPs) have not been fully established, especially when applied to rare and quantity-limited biological samples. We previously demonstrated that a mixture containing 1-butyl-3-methylimidazolium cyanate, an ionic liquid (IL), and NaOH (termed i-soln) is an excellent solubilizer for insoluble aggregates. In this study, we present a combined i-soln-assisted proteomic sample preparation platform (termed pTRUST), which is compatible with starting materials in the sub-microgram range, using our previously reported i-soln-based sample preparation strategy (iBOPs) and an in-StageTip technique. This novel and straightforward approach allows for the rapid solubilization and processing of a variety of IMPs from human samples to support highly sensitive mass spectrometry analysis. We also demonstrated that the performance of this technology surpasses that of conventional methods such as filter-aided sample preparation methods, FASP and i-FASP. The convenience and availability of pTRUST technology using the IL system have great potential for proteomic identification and characterization of novel drug targets and disease biology in research and clinical settings.
Asunto(s)
Líquidos Iónicos , Proteoma , Humanos , Proteoma/metabolismo , Proteómica/métodos , Cromatografía Liquida/métodos , Proteínas de la Membrana/metabolismoRESUMEN
Feeder cells and the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) in a culture medium promote mitosis and cell division in cultured cells. These are also added to nutrient medium for the cultivation of highly active in mitosis and dividing zygotes, produced in vitro or isolated from pollinated ovaries. In the study, an in vitro fertilization (IVF) system was used to study the precise effects of feeder cells and 2,4-D on the growth and development of rice (Oryza sativa L.) zygote. The elimination of 2,4-D from the culture medium did not affect the early developmental profiles of the zygotes, but decreased the division rates of multicellular embryos. The omission of feeder cells resulted in defective karyogamy, fusion between male and female nuclei, and the subsequent first division of the cultured zygotes. The culture of zygotes in a conditioned medium corrected developmental disorders. Proteome analyses of the conditioned medium revealed the presence of abundant hydrolases possibly released from the feeder cells. Exogenously applied α-amylase ameliorated karyogamy and promoted zygote development. It is suggested that hydrolytic enzymes, including α-amylase, released from feeder cells may be involved in the progression of zygotic development.
Asunto(s)
Oryza , Cigoto , Medios de Cultivo Condicionados/farmacología , Mitosis , Fertilización In Vitro , Células Cultivadas , alfa-Amilasas , Ácido 2,4-DiclorofenoxiacéticoRESUMEN
Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.
Asunto(s)
Euglena gracilis/química , ARN Ribosómico/química , Ribosomas/química , Microscopía por Crioelectrón , Citoplasma/química , Euglena gracilis/genética , Euglena gracilis/metabolismo , Modelos Moleculares , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/químicaRESUMEN
TDP-43 regulates cellular levels of Cajal bodies (CBs) that provide platforms for the assembly and RNA modifications of small nuclear ribonucleoproteins (snRNPs) involved in pre-mRNA splicing. Alterations in these snRNPs may be linked to pathogenesis of amyotrophic lateral sclerosis. However, specific roles for TDP-43 in CBs remain unknown. Here, we demonstrate that TDP-43 regulates the CB localization of four UG-rich motif-bearing C/D-box-containing small Cajal body-specific RNAs (C/D scaRNAs; i.e. scaRNA2, 7, 9 and 28) through the direct binding to these scaRNAs. TDP-43 enhances binding of a CB-localizing protein, WD40-repeat protein 79 (WDR79), to a subpopulation of scaRNA2 and scaRNA28; the remaining population of the four C/D scaRNAs was localized to CB-like structures even with WDR79 depletion. Depletion of TDP-43, in contrast, shifted the localization of these C/D scaRNAs, mainly into the nucleolus, as well as destabilizing scaRNA2, and reduced the site-specific 2'-O-methylation of U1 and U2 snRNAs, including at 70A in U1 snRNA and, 19G, 25G, 47U and 61C in U2 snRNA. Collectively, we suggest that TDP-43 and WDR79 have separate roles in determining CB localization of subsets of C/D and H/ACA scaRNAs.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Cuerpos Enrollados/genética , Proteínas de Unión al ADN/genética , Proteínas/genética , Esclerosis Amiotrófica Lateral/patología , Nucléolo Celular/genética , Cuerpos Enrollados/metabolismo , Citidina/análogos & derivados , Citidina/genética , Células HeLa , Humanos , Chaperonas Moleculares , ARN Guía de Kinetoplastida/genética , ARN Nuclear Pequeño/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , TelomerasaRESUMEN
Pseudouridine (Ψ) is the only "mass-silent" nucleoside produced by post-transcriptional RNA modification. We developed a mass spectrometry (MS)-based technique coupled with in vivo deuterium (D) labeling of uridines for direct determination of Ψs in cellular RNA and applied it to the comprehensive analysis of post-transcriptional modifications in human ribosomal RNAs. The method utilizes human TK6/mouse FM3A cells deficient in uridine monophosphate synthase using a CRISPR-Cas9 technique to turn off de novo uridine synthesis and fully labels uridines with D at uracil positions 5 and 6 by cultivating the cells in a medium containing uridine-5,6-D2. The pseudouridylation reaction in those cells results in the exchange of the D at the C5 of uracil with hydrogen from solvent, which produces a -1 Da mass shift, thus allowing MS-based determination of RNA Ψs. We present here the experimental details of this method and show that it allows the identification of all Ψs in human major nuclear and nucleolar RNAs, including several previously unknown Ψs. Because the method allows direct determination of Ψs at the femtomole level of RNA, it will serve as a useful tool for structure/function studies of a wide variety of noncoding RNAs.
Asunto(s)
Seudouridina/análisis , Procesamiento Postranscripcional del ARN , ARN Ribosómico/análisis , ARN Ribosómico/metabolismo , ARN Nuclear Pequeño/análisis , ARN Nuclear Pequeño/metabolismo , Animales , Línea Celular , Deuterio/química , Humanos , Marcaje Isotópico , Espectrometría de Masas , Ratones , Complejos Multienzimáticos/química , Orotato Fosforribosiltransferasa/química , Orotidina-5'-Fosfato Descarboxilasa/química , Seudouridina/química , ARN Ribosómico/química , ARN Nuclear Pequeño/químicaRESUMEN
RATIONALE: Multilineage-differentiating stress enduring (Muse) cells, pluripotent marker stage-specific embryonic antigen-3+ cells, are nontumorigenic endogenous pluripotent-like stem cells obtainable from various tissues including the bone marrow. Their therapeutic efficiency has not been validated in acute myocardial infarction. OBJECTIVE: The main objective of this study is to clarify the efficiency of intravenously infused rabbit autograft, allograft, and xenograft (human) bone marrow-Muse cells in a rabbit acute myocardial infarction model and their mechanisms of tissue repair. METHODS AND RESULTS: In vivo dynamics of Nano-lantern-labeled Muse cells showed preferential homing of the cells to the postinfarct heart at 3 days and 2 weeks, with ≈14.5% of injected GFP (green fluorescent protein)-Muse cells estimated to be engrafted into the heart at 3 days. The migration and homing of the Muse cells was confirmed pharmacologically (S1PR2 [sphingosine monophosphate receptor 2]-specific antagonist JTE-013 coinjection) and genetically (S1PR2-siRNA [small interfering ribonucleic acid]-introduced Muse cells) to be mediated through the S1P (sphingosine monophosphate)-S1PR2 axis. They spontaneously differentiated into cells positive for cardiac markers, such as cardiac troponin-I, sarcomeric α-actinin, and connexin-43, and vascular markers. GCaMP3 (GFP-based Ca calmodulin probe)-labeled Muse cells that engrafted into the ischemic region exhibited increased GCaMP3 fluorescence during systole and decreased fluorescence during diastole. Infarct size was reduced by ≈52%, and the ejection fraction was increased by ≈38% compared with vehicle injection at 2 months, ≈2.5 and ≈2.1 times higher, respectively, than that induced by mesenchymal stem cells. These effects were partially attenuated by the administration of GATA4-gene-silenced Muse cells. Muse cell allografts and xenografts efficiently engrafted and recovered functions, and allografts remained in the tissue and sustained functional recovery for up to 6 months without immunosuppression. CONCLUSIONS: Muse cells may provide reparative effects and robust functional recovery and may, thus, provide a novel strategy for the treatment of acute myocardial infarction.
Asunto(s)
Lisofosfolípidos/fisiología , Infarto del Miocardio/cirugía , Células Madre Pluripotentes/trasplante , Receptores de Lisoesfingolípidos/fisiología , Esfingosina/análogos & derivados , Aloinjertos , Animales , Autoinjertos , Diferenciación Celular , Movimiento Celular/fisiología , Factor de Transcripción GATA4/antagonistas & inhibidores , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/fisiología , Supervivencia de Injerto , Proteínas Fluorescentes Verdes/análisis , Xenoinjertos , Humanos , Luciferasas/análisis , Proteínas Luminiscentes/análisis , Masculino , Infarto del Miocardio/patología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Conejos , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/genética , Proteínas Recombinantes de Fusión/análisis , Especificidad de la Especie , Esfingosina/fisiología , Receptores de Esfingosina-1-FosfatoRESUMEN
During ribosome biogenesis, ribosomal RNAs acquire various chemical modifications that ensure the fidelity of translation, and dysregulation of the modification processes can cause proteome changes as observed in cancer and inherited human disorders. Here, we report the complete chemical modifications of all RNAs of the human 80S ribosome as determined with quantitative mass spectrometry. We assigned 228 sites with 14 different post-transcriptional modifications, most of which are located in functional regions of the ribosome. All modifications detected are typical of eukaryotic ribosomal RNAs, and no human-specific modifications were observed, in contrast to a recently reported cryo-electron microscopy analysis. While human ribosomal RNAs appeared to have little polymorphism regarding the post-transcriptional modifications, we found that pseudouridylation at two specific sites in 28S ribosomal RNA are significantly reduced in ribosomes of patients with familial dyskeratosis congenita, a genetic disease caused by a point mutation in the pseudouridine synthase gene DKC1. The landscape of the entire epitranscriptomic ribosomal RNA modifications provides a firm basis for understanding ribosome function and dysfunction associated with human disease.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Ribosómico 28S/genética , ARN/genética , Ribosomas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Transformada , Microscopía por Crioelectrón , Disqueratosis Congénita/genética , Células HeLa , Humanos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , Seudouridina/metabolismo , ARN/química , ARN/metabolismo , ARN Ribosómico 28S/química , ARN Ribosómico 28S/metabolismo , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.
Asunto(s)
Cromatina/genética , Sitios Frágiles del Cromosoma/genética , Replicación del ADN/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Inestabilidad Genómica/genética , Afidicolina/farmacología , Línea Celular Tumoral , Cromatina/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , Daño del ADN , Reparación del ADN , Replicación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Conformación de Ácido Nucleico , Transducción de Señal/genética , Ubiquitinación/efectos de los fármacosRESUMEN
Exocytosis of secretory granules entails budding from the trans-Golgi network, sorting and maturation of cargo proteins, and trafficking and fusion to the plasma membrane. Rab27a regulates the late steps in this process, such as granule recruitment to the fusion site, whereas Rab2a functions in the early steps, such as granule biogenesis and maturation. Here, we demonstrate that these two small GTPases simultaneously bind to Noc2 (also known as RPH3AL) in a GTP-dependent manner, although Rab2a binds only after Rab27a has bound. In pancreatic ß-cells, the ternary Rab2a-Noc2-Rab27a complex specifically localizes on perinuclear immature granules, whereas the binary Noc2-Rab27a complex localizes on peripheral mature granules. In contrast to the wild type, Noc2 mutants defective in binding to Rab2a or Rab27a fail to promote glucose-stimulated insulin secretion. Although knockdown of any component of the ternary complex markedly inhibits insulin secretion, only knockdown of Rab2a or Noc2, and not that of Rab27a, impairs cargo processing from proinsulin to insulin. These results suggest that the dual effector, Noc2, regulates the transition from Rab2a-mediated granule biogenesis to Rab27a-mediated granule exocytosis.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Exocitosis , Proteínas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteínas Mutantes/metabolismo , Unión Proteica , Proteínas/química , Ratas , Proteínas rab27 de Unión a GTPRESUMEN
A wide variety of proteomic methods have been applied for protein profiling of insoluble aggregates or inclusion bodies deposited in various cells or tissues. However, these are essentially optimized or modified classical protein chemistry techniques using conventional denaturing agents such as formic acid, urea, and sodium dodecyl sulfate (SDS). The use of these denaturants has several shortcomings, including limited solubilization, contamination, and restrictions on absolute sample quantity and throughput. Here, we describe an alternative proteomic sample preparation platform for widespread aggregation analysis. This approach combines two techniques, (1) the use of ionic liquid for protein solubilization and (2) the recently published microbead-based and organic-media-assisted proteolysis strategy (BOPs), into a single-tube workflow. We demonstrate that the combined approach (iBOPs) enabled the successful solubilization of heat-aggregated hen egg whites within 10 min and supported sensitive mass spectrometry (MS) analysis. The performance of the iBOPs system surpassed those of conventional detergents and chaotropes. Moreover, this technology enabled ultrasensitive proteomic characterization of protein aggregates deposited in individual Caenorhabditis elegans nematodes. We identified ubiquitin and other molecules as candidate stochastic factors whose accumulation levels varied among aging nematode individuals. The sensitivity and applicability of the present iBOPs make it especially attractive for next-stage aggregate proteomic analysis of various biological processes.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas del Huevo/química , Líquidos Iónicos , Agregado de Proteínas , Proteínas/química , Proteómica/métodos , Animales , ImidazolesRESUMEN
RNA post-transcriptional modifications are common in all kingdoms of life and are predominantly affiliated with methylations at various nucleobase positions. Methylations occur frequently at specific sites on the RNA nucleobases and appear to regulate site-specific intermolecular/intramolecular interactions. Herein, we present a method that utilizes liquid chromatography-mass spectrometry (LC-MS) to identify positional monomethylated RNA nucleoside isomers. The method produces profiles of in-source fragmentation and subsequent tandem mass spectrometry (MS2) (pseudo-MS3) of RNase-digested fragments of an RNA and distinguishes between positional methylated nucleobase isomers by comparing their intranucleobase fragment ion profiles with signature profiles derived from authentic isomers. For method validation, we independently determined the positions of all known monomethylated nucleoside isomers in the Escherichia coli 16S/23S rRNAs. As proof of concept, we further applied this technology to fully characterize the base-modified nucleoside positional isomers, in rRNAs derived from Leishmania donovani, a human blood parasite afflicting millions around the globe. The method described herein will be highly beneficial for the delineation of RNA modification profiles in various cellular RNAs, and as it only requires a subpicomole amount of RNA, it could also be used for the structure-function studies of RNA populations represented in minute amounts in the cell.
Asunto(s)
Escherichia coli/genética , Leishmania/genética , Nucleósidos/análisis , ARN Ribosómico 18S/análisis , ARN Ribosómico/análisis , Humanos , Metilación , Nucleósidos/química , Procesamiento Postranscripcional del ARN , ARN Ribosómico/química , ARN Ribosómico 18S/químicaRESUMEN
Phototropin1 (phot1) perceives low- to high-fluence blue light stimuli and mediates both the first and second positive phototropisms. High-fluence blue light is known to induce autophosphorylation of phot1, leading to the second positive phototropism. However, the phosphorylation status of phot1 by low-fluence blue light that induces the first positive phototropism had not been observed. Here, we conducted a phosphoproteomic analysis of maize coleoptiles to investigate the fluence-dependent phosphorylation status of Zmphot1. High-fluence blue light induced phosphorylation of Zmphot1 at several sites. Notably, low-fluence blue light significantly increased the phosphorylation level of Ser291 in Zmphot1. Furthermore, Ser291-phosphorylated and Ser369Ser376-diphosphorylated peptides were found to be more abundant in the low-fluence blue light-irradiated sides than in the shaded sides of coleoptiles. The roles of these phosphorylation events in phototropism were explored by heterologous expression of ZmPHOT1 in the Arabidopsis thaliana phot1phot2 mutant. The first positive phototropism was restored in wild-type ZmPHOT1-expressing plants; however, plants expressing S291A-ZmPHOT1 or S369AS376A-ZmPHOT1 showed significantly reduced complementation rates. All transgenic plants tested in this study exhibited a normal second positive phototropism. These findings provide the first indication that low-fluence blue light induces phosphorylation of Zmphot1 and that this induced phosphorylation is crucial for the first positive phototropism.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Luz , Fototropismo/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Fosforilación/genética , Fosforilación/efectos de la radiación , Fototropismo/genética , Fototropismo/efectos de la radiación , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Zea mays/genética , Zea mays/metabolismo , Zea mays/efectos de la radiaciónRESUMEN
Ribosome biogenesis occurs successively in the nucleolus, nucleoplasm, and cytoplasm. Maturation of the ribosomal small subunit is completed in the cytoplasm by incorporation of a particular class of ribosomal proteins and final cleavage of 18S-E pre-rRNA (18S-E). Here, we show that poly(A)-specific ribonuclease (PARN) participates in steps leading to 18S-E maturation in human cells. We found PARN as a novel component of the pre-40S particle pulled down with the pre-ribosome factor LTV1 or Bystin. Reverse pull-down analysis revealed that PARN is a constitutive component of the Bystin-associated pre-40S particle. Knockdown of PARN or exogenous expression of an enzyme-dead PARN mutant (D28A) accumulated 18S-E in both the cytoplasm and nucleus. Moreover, expression of D28A accumulated 18S-E in Bystin-associated pre-40S particles, suggesting that the enzymatic activity of PARN is necessary for the release of 18S-E from Bystin-associated pre-40S particles. Finally, RNase H-based fragmentation analysis and 3Î-sequence analysis of 18S-E species present in cells expressing wild-type PARN or D28A suggested that PARN degrades the extended regions encompassing nucleotides 5-44 at the 3Î end of mature 18S rRNA. Our results reveal a novel role for PARN in ribosome biogenesis in human cells.
Asunto(s)
Exorribonucleasas/fisiología , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico 18S/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Moléculas de Adhesión Celular/análisis , Exorribonucleasas/análisis , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Células HeLa , Humanos , Mutación , Proteínas Ribosómicas/análisis , Subunidades Ribosómicas Pequeñas de Eucariotas/químicaRESUMEN
During the biogenesis of U1 small nuclear ribonucleoprotein, a small population of U1 snRNA molecules acquires an extra methylation at the first transcribed nucleotide and a nucleolytic cleavage to remove the 3' structured region including the Sm protein-binding site and stem-loop 4. These modifications occur before hypermethylation of the monomethylated 5' cap, whereby producing truncated forms of U1 snRNA (U1-tfs) that are diverted from the normal pathway to a processing body-associated degradation pathway. Here, we demonstrate that a small population of U2 snRNA molecules receives post-transcriptional modifications similar to those of U1 to yield U2-tfs. Like U1-tfs, U2-tfs molecules were produced from transcripts of the U2 snRNA gene having all cis-elements or lacking the 3' box. Unlike U1-tfs, however, a portion of U2-tfs received additional uridylylation of up to 5 nucleotides in length at position 87 (designated as U2-tfs-polyU) and formed an Sm protein-binding site-like structure that was stabilized by the small nuclear ribonucleoprotein SmB/B' probably as a part of heptameric Sm core complex that associates to the RNA. Both U2-tfs and U2-tfs-polyU were degraded by a nuclease distinct from the canonical Dis3L2 by a process catalyzed by terminal uridylyltransferase 7. Collectively, our data suggest that U2 snRNA biogenesis is regulated, at least in part, by a novel degradation pathway to ensure that defective U2 molecules are not incorporated into the spliceosome.
Asunto(s)
ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Línea Celular , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , ARN Nuclear Pequeño/genética , Ribonucleoproteínas Nucleares Pequeñas/genéticaRESUMEN
We present the complete chemical structures of the rRNAs from the eukaryotic model organism, Saccharomyces cerevisiae The final structures, as determined with mass spectrometry-based methodology that includes a stable isotope-labelled, non-modified reference RNA, contain 112 sites with 12 different post-transcriptional modifications, including a previously unidentified pseudouridine at position 2345 in 25S rRNA. Quantitative mass spectrometry-based stoichiometric analysis of the different modifications at each site indicated that 94 sites were almost fully modified, whereas the remaining 18 sites were modified to a lesser extent. Superimposed three-dimensional modification maps for S. cerevisiae and Schizosaccharomyces pombe rRNAs confirmed that most of the modified nucleotides are located in functionally important interior regions of the ribosomes. We identified snR9 as the snoRNA responsible for pseudouridylation of U2345 and showed that this pseudouridylation occurs co-transcriptionally and competitively with 2'-O-methylation of U2345. This study ends the uncertainty concerning whether all modified nucleotides in S. cerevisiae rRNAs have been identified and provides a resource for future structural, functional and biogenesis studies of the eukaryotic ribosome.
Asunto(s)
ARN de Hongos/química , ARN de Hongos/genética , ARN Ribosómico/química , ARN Ribosómico/genética , Saccharomyces cerevisiae/genética , Composición de Base , Cromatografía Liquida , Espectrometría de Masas , Metilación , Modelos Moleculares , Conformación Molecular , Conformación de Ácido Nucleico , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , Ribosomas/química , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Pseudouridine (5-ribosyluracil, Ψ) is the only 'mass-silent' nucleoside produced by post-transcriptional RNA modification. We describe here a novel mass spectrometry (MS)-based method for direct determination of Ψ in RNA. The method assigns a Ψ-containing nucleolytic RNA fragment by an accurate measurement of a signature doubly dehydrated nucleoside anion ([C9H7N2O4](1-),m/z207.04) produced by collision-induced dissociation MS, and it determines the Ψ-containing nucleotide sequence by pseudo-MS(3), i.e. in-source fragmentation followed by MS(2) By applying this method, we identified all of the known Ψs in the canonical human spliceosomal snRNAs and, unexpectedly, found two previously unknown Ψs in the U5 and U6 snRNAs. Because the method allows direct determination of Ψ in a subpicomole quantity of RNA, it will serve as a useful tool for the structure/function studies of a wide variety of non-coding RNAs.