RESUMO
Telomere length is associated with the proliferative potential of cells. Telomerase is an enzyme that elongates telomeres throughout the entire lifespan of an organism in stem cells, germ cells, and cells of constantly renewed tissues. It is activated during cellular division, including regeneration and immune responses. The biogenesis of telomerase components and their assembly and functional localization to the telomere is a complex system regulated at multiple levels, where each step must be tuned to the cellular requirements. Any defect in the function or localization of the components of the telomerase biogenesis and functional system will affect the maintenance of telomere length, which is critical to the processes of regeneration, immune response, embryonic development, and cancer progression. An understanding of the regulatory mechanisms of telomerase biogenesis and activity is necessary for the development of approaches toward manipulating telomerase to influence these processes. The present review focuses on the molecular mechanisms involved in the major steps of telomerase regulation and the role of post-transcriptional and post-translational modifications in telomerase biogenesis and function in yeast and vertebrates.
Assuntos
Neoplasias , Telomerase , Animais , Humanos , Telomerase/metabolismo , Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Mammalian mitochondrial ribosomes contain a set of modified nucleotides, which is distinct from that of the cytosolic ribosomes. Nucleotide m4C840 of the murine mitochondrial 12S rRNA is equivalent to the dimethylated m4Cm1402 residue of Escherichia coli 16S rRNA. Here we demonstrate that mouse METTL15 protein is responsible for the formation of m4C residue of the 12S rRNA. Inactivation of Mettl15 gene in murine cell line perturbs the composition of mitochondrial protein biosynthesis machinery. Identification of METTL15 interaction partners revealed that the likely substrate for this RNA methyltransferase is an assembly intermediate of the mitochondrial small ribosomal subunit containing an assembly factor RBFA.
Assuntos
Metiltransferases/metabolismo , Mitocôndrias/enzimologia , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/enzimologia , Animais , Células Cultivadas , Metilação , Camundongos , Mitocôndrias/metabolismo , RNA Mitocondrial/química , RNA Mitocondrial/metabolismo , RNA Ribossômico/química , RNA Ribossômico 28S/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/metabolismoRESUMO
In bacteria, rapid adaptation to changing environmental conditions depends on the interplay between housekeeping and alternative σ factors, responsible for transcription of specific regulons by RNA polymerase (RNAP). In comparison with alternative σ factors, primary σs contain poorly conserved region 1.1, whose functions in transcription are only partially understood. We found that a single mutation in region 1.1 in Escherichia coli σ70 rewires transcription regulation during cell growth resulting in profound phenotypic changes. Despite its destabilizing effect on promoter complexes, this mutation increases the activity of rRNA promoters and also decreases RNAP sensitivity to the major regulator of stringent response DksA. Using total RNA sequencing combined with single-cell analysis of gene expression we showed that changes in region 1.1 disrupt the balance between the "greed" and "fear" strategies thus making the cells more susceptible to environmental threats and antibiotics. Our results reveal an unexpected role of σ region 1.1 in growth-dependent transcription regulation and suggest that changes in this region may facilitate rapid switching of RNAP properties in evolving bacterial populations.
Assuntos
Divisão Celular , Regulação Bacteriana da Expressão Gênica , Fator sigma/genética , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação Puntual , Domínios Proteicos , Fator sigma/química , Fator sigma/metabolismo , Transcrição GênicaRESUMO
First triplets of mRNA coding region affect the yield of translation. We have applied the flowseq method to analyze >30 000 variants of the codons 2-11 of the fluorescent protein reporter to identify factors affecting the protein synthesis. While the negative influence of mRNA secondary structure on translation has been confirmed, a positive role of rare codons at the beginning of a coding sequence for gene expression has not been observed. The identity of triplets proximal to the start codon contributes more to the protein yield then more distant ones. Additional in-frame start codons enhance translation, while Shine-Dalgarno-like motifs downstream the initiation codon are inhibitory. The metabolic cost of amino acids affects the yield of protein in the poor medium. The most efficient translation was observed for variants with features resembling those of native Escherichia coli genes.
Assuntos
Códon de Iniciação/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/genética , Códon de Iniciação/ultraestrutura , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/ultraestrutura , Ribossomos/genética , Ribossomos/ultraestruturaRESUMO
Genes coding for small peptides have been frequently misannotated as long noncoding RNA (lncRNA) genes. Here we have demonstrated that one such transcript is translated into a 56-amino-acid-long peptide conserved in chordates, corroborating the work published while this manuscript was under review. The Mtln peptide could be detected in mitochondria of mouse cell lines and tissues. In line with its mitochondrial localization, lack of the Mtln decreases the activity of mitochondrial respiratory chain complex I. Unlike the integral components and assembly factors of NADH:ubiquinone oxidoreductase, Mtln does not alter its enzymatic activity directly. Interaction of Mtln with NADH-dependent cytochrome b5 reductase stimulates complex I functioning most likely by providing a favorable lipid composition of the membrane. Study of Mtln illuminates the importance of small peptides, whose genes might frequently be misannotated as lncRNAs, for the control of vitally important cellular processes.
Assuntos
Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Peptídeos/metabolismo , RNA Longo não Codificante/metabolismo , Sequência de Aminoácidos , Animais , Respiração Celular , Citosol/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Camundongos , NAD/metabolismo , Células NIH 3T3 , Consumo de Oxigênio , Fosfolipídeos/metabolismo , RNA Longo não Codificante/genética , Triglicerídeos/metabolismoRESUMO
Following nanoarchitectural approach, mesoporous halloysite nanotubes with internal surface composed of alumina were loaded with 5-6 nm RuCo nanoparticles by sequential loading/reduction procedure. Ruthenium nanoclusters were loaded inside clay tube by microwave-assisted method followed by cobalt ions electrostatic attraction to ruthenium during wetness impregnation step. Developed nanoreactors with bimetallic RuCo nanoparticles were investigated as catalysts for the Fischer-Tropsch process. The catalyst with 14.3 wt.% of Co and 0.15 wt.% of Ru showed high activity (СO conversion reached 24.6%), low selectivity to methane (11.9%), CO2 (0.3%), selectivity to C5+ hydrocarbons of 79.1% and chain growth index (α) = 0.853. Proposed nanoreactors showed better selectivity to target products combined with high activity in comparison to the similar bimetallic systems supported on synthetic porous materials. It was shown that reducing agent (NaBH4 or H2) used to obtain Ru nanoclusters at first synthesis step played a very important role in the reducibility and selectivity of resulting RuCo catalysts.
RESUMO
Flow-seq is a method that combines fluorescently activated cell sorting and next-generation sequencing to deduce a large amount of data about translation efficiency from a single experiment. Here, we constructed a library of fluorescent protein-based reporters preceded by a set of 648 natural 5'-untranslated regions (5'-UTRs) of Escherichia coli genes. Usually, Flow-seq libraries are constructed using uniform-length sequence elements, in contrast to natural situations, where functional elements are of heterogenous lengths. Here, we demonstrated that a 5'-UTR library of variable length could be created and analyzed with Flow-seq. In line with previous Flow-seq experiments with randomized 5'-UTRs, we observed the influence of an RNA secondary structure and Shine-Dalgarno sequences on translation efficiency; however, the variability of these parameters for natural 5'-UTRs in our library was smaller in comparison with randomized libraries. In line with this, we only observed a 30-fold difference in translation efficiency between the best and worst bins sorted with this factor. The results correlated with those obtained with ribosome profiling.
Assuntos
Escherichia coli , Ribossomos , Escherichia coli/genética , Escherichia coli/metabolismo , Regiões 5' não Traduzidas/genética , Ribossomos/genética , Ribossomos/metabolismo , Biblioteca Gênica , Biossíntese de ProteínasRESUMO
The biodiversity of microorganisms is maintained by intricate nets of interactions between competing species. Impaired functionality of human microbiomes correlates with their reduced biodiversity originating from aseptic environmental conditions and antibiotic use. Microbiomes of wild animals are free of these selective pressures. Microbiota provides a protecting shield from invasion by pathogens in the wild, outcompeting their growth in specific ecological niches. We applied ultrahigh-throughput microfluidic technologies for functional profiling of microbiomes of wild animals, including the skin beetle, Siberian lynx, common raccoon dog, and East Siberian brown bear. Single-cell screening of the most efficient killers of the common human pathogen Staphylococcus aureus resulted in repeated isolation of Bacillus pumilus strains. While isolated strains had different phenotypes, all of them displayed a similar set of biosynthetic gene clusters (BGCs) encoding antibiotic amicoumacin, siderophore bacillibactin, and putative analogs of antimicrobials including bacilysin, surfactin, desferrioxamine, and class IId cyclical bacteriocin. Amicoumacin A (Ami) was identified as a major antibacterial metabolite of these strains mediating their antagonistic activity. Genome mining indicates that Ami BGCs with this architecture subdivide into three distinct families, characteristic of the B. pumilus, B. subtilis, and Paenibacillus species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated B. pumilus strains efficiently inhibit the growth of both Gram-positive S. aureus and Gram-negative E. coli in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing B. pumilus can be attributed to the metabolomic profile predetermined by their biosynthetic fingerprint. Ultrahigh-throughput isolation of natural probiotic strains from wild animal microbiomes, as well as their metabolic reprogramming, opens up a new avenue for pathogen control and microbiome remodeling in the food industry, agriculture, and healthcare.
Assuntos
Animais Selvagens/microbiologia , Antibacterianos/administração & dosagem , Bacillus pumilus/química , Escherichia coli/crescimento & desenvolvimento , Microbiota , Probióticos/administração & dosagem , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , Genoma Bacteriano , Metaboloma , Família Multigênica , Probióticos/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacosRESUMO
Cell functioning is tightly regulated process. For many years, research in the fields of proteomics and functional genomics has been focused on the role of proteins in cell functioning. The advances in science have led to the uncovering that short open reading frames, previously considered non-functional, serve a variety of functions. Short reading frames in polycistronic mRNAs often regulate their stability and translational efficiency of the main reading frame. The improvement of proteomic analysis methods has made it possible to identify the products of translation of short open reading frames in quantities that suggest the existence of functional role of those peptides and short proteins. Studies demonstrating their role unravel a new level of the regulation of cell functioning and its adaptation to changing conditions. This review is devoted to the analysis of functions of recently discovered peptides and short proteins.
Assuntos
Peptídeos/metabolismo , Proteínas/metabolismo , Autofagia , Reparo do DNA , Humanos , Mitocôndrias/metabolismo , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismoRESUMO
Microbiome spectra serve as critical clues to elucidate the evolutionary biology pathways, potential pathologies, and even behavioral patterns of the host organisms. Furthermore, exotic sources of microbiota represent an unexplored niche to discover microbial secondary metabolites. However, establishing the bacterial functionality is complicated by an intricate web of interactions inside the microbiome. Here we apply an ultrahigh-throughput (uHT) microfluidic droplet platform for activity profiling of the entire oral microbial community of the Siberian bear to isolate Bacillus strains demonstrating antimicrobial activity against Staphylococcus aureus Genome mining allowed us to identify antibiotic amicoumacin A (Ami) as responsible for inhibiting the growth of S. aureus Proteomics and metabolomics revealed a unique mechanism of Bacillus self-resistance to Ami, based on a subtle equilibrium of its deactivation and activation by kinase AmiN and phosphatase AmiO, respectively. We developed uHT quantitative single-cell analysis to estimate antibiotic efficacy toward different microbiomes and used it to determine the activity spectra of Ami toward human and Siberian bear microbiota. Thus, uHT microfluidic droplet platform activity profiling is a powerful tool for discovering antibiotics and quantifying external influences on a microbiome.
Assuntos
Antibacterianos/farmacologia , Cumarínicos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Metabolômica/métodos , Animais , Antibacterianos/metabolismo , Bacillus pumilus/efeitos dos fármacos , Bacillus pumilus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cumarínicos/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Farmacorresistência Bacteriana/fisiologia , Microbioma Gastrointestinal/fisiologia , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Dispositivos Lab-On-A-Chip , Proteômica/métodos , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Análise de Célula Única/métodos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Ursidae/microbiologiaRESUMO
RNA molecules of all species contain modified nucleotides and particularly m5U residues. The vertebrate mitochondrial small subunit rRNA contains m5U nucleotide in a unique site. In this work we found an enzyme, TRMT2B, responsible for the formation of this nucleotide and m5U residues in a number of mitochondrial tRNA species. Inactivation of the Trmt2B gene leads to a reduction of the activity of respiratory chain complexes I, III and IV, containing the subunits synthesized by the mitochondrial protein synthesis apparatus. Comparative sequence analysis of m5U-specific RNA methyltransferases revealed an unusual evolutionary pathway of TRMT2B formation which includes consecutive substrate specificity switches from the large subunit rRNA to tRNA and then to the small subunit rRNA.
Assuntos
Mitocôndrias/enzimologia , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , tRNA Metiltransferases/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Metilação , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Conformação de Ácido Nucleico , RNA Mitocondrial/química , RNA Mitocondrial/metabolismo , RNA Ribossômico/química , RNA de Transferência/química , Especificidade por Substrato , Timina/metabolismo , tRNA Metiltransferases/genéticaRESUMO
Several studies have described functional peptides encoded in RNA that are considered to be noncoding. Telomerase RNA together with telomerase reverse transcriptase and regulatory proteins make up the telomerase complex, the major component of the telomere length-maintaining machinery. In contrast to protein subunits, telomerase RNA is expressed constitutively in most somatic cells where telomerase reverse transcriptase is absent. We show here that the transcript of human telomerase RNA codes a 121 amino acid protein (hTERP). The existence of hTERP was shown by immunoblotting, immunofluorescence microscopy and mass spectroscopy. Gain-of-function and loss-of-function experiments showed that hTERP protects cells from drug-induced apoptosis and participates in the processing of autophagosome. We suggest that hTERP regulates crosstalk between autophagy and apoptosis and is involved in cellular adaptation under stress conditions.
Assuntos
Adaptação Fisiológica/genética , Apoptose/genética , Autofagia/genética , RNA Mensageiro/genética , RNA/genética , Telomerase/genética , Telômero/metabolismo , Sequência de Aminoácidos , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Gatos , Linhagem Celular Tumoral , Clonagem Molecular , Doxorrubicina/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Cavalos , Humanos , Células Jurkat , Camundongos , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Estresse Fisiológico , Telomerase/metabolismo , Telômero/química , Homeostase do TelômeroRESUMO
Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.
Assuntos
Butirilcolinesterase/química , Ensaios de Triagem em Larga Escala/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Paraoxon/química , Análise de Célula Única/instrumentação , Antibiose , Biodiversidade , Comunicação Celular , Emulsões , Citometria de Fluxo , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Óleos Voláteis/química , Fenótipo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Água/químicaRESUMO
Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5Î untranslated regions (5Î-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5Î-UTR sequences and introduced random and designed mutations into natural and artificially selected 5Î-UTRs. Several distinct properties could be ascribed to a group of 5Î-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5Î-UTRs.
Assuntos
Regiões 5' não Traduzidas , Escherichia coli/genética , Biossíntese de Proteínas , Sequências Reguladoras de Ácido Ribonucleico , Separação Celular , Citometria de Fluxo , Genes Reporter , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Conformação de Ácido Nucleico , Nucleotídeos/fisiologiaRESUMO
BACKGROUND: In the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity. RESULTS: Mutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones. CONCLUSIONS: Comparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.
Assuntos
Genômica , Pichia/enzimologia , Pichia/genética , Telomerase/deficiência , Termotolerância , Transcrição Gênica , Autofagia/genética , Metabolismo dos Carboidratos/genética , Dano ao DNA/genética , Metabolismo Energético/genética , Meio Ambiente , Genes Fúngicos/genética , Espaço Intracelular/metabolismo , Pichia/citologia , Pichia/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética , Encurtamento do Telômero/genéticaRESUMO
Telomerase is a key participant in the telomere length maintaining system in eukaryotic cells. Telomerase RNA and protein reverse transcriptase subunits are essential for the appearance of active telomerase in vitro. Telomerase is active in many cancer types and is a potential target for anticancer drug development. Here we report a new approach for impairing telomerase function at the stage of human telomerase assembly. The approach is based on the application of chimeric bifunctional oligonucleotides that contain two oligonucleotide parts complementary to the functional domains of telomerase RNA connected with non-nucleotide linkers in different orientations (5'-3', 5'-5' or 3'-3'). Such chimeras inhibited telomerase in vitro in the nM range, but were effective in vivo in sub-nM concentrations, predominantly due to their effect on telomerase assembly and dimerization.
Assuntos
Oligonucleotídeos/química , Telomerase/antagonistas & inibidores , Dimerização , Células HEK293 , Humanos , RNA/química , RNA/metabolismo , Telomerase/química , Telomerase/metabolismoRESUMO
Despite the mechanical and physical properties of polyurethane foams (PUF), their application is still hindered by high inflammability. The elaboration of effective, low-cost, and environmentally friendly fire retardants remains a pressing issue that must be addressed. This work aims to show the feasibility of the successful application of natural nanomaterials, such as halloysite nanotubes and nanocellulose, as promising additives to the commercial halogen-free, fire-retardant triphenyl phosphate (TPP) to enhance the flame retardance of open-cell polyurethane foams. The nanocomposite foams were synthesized by in situ polymerization. Investigation of the mechanical properties of the nanocomposite PUF revealed that the nanoscale additives led to a notable decrease in the foam's compressibility. The obtained results of the flammability tests clearly indicate that there is a prominent synergetic effect between the fire-retardant and the natural nanoscale additives. The nanocomposite foams containing a mixture of TPP (10 and 20 parts per hundred polyol by weight) and either 10 wt.% of nanocellulose or 20 wt.% of halloysite demonstrated the lowest burning rate without dripping and were rated as HB materials according to UL 94 classification.
RESUMO
Ribosome biogenesis is essential for the functioning of living cells. In higher eukaryotes, this multistep process is tightly controlled and involves a variety of specialized proteins and RNAs. This pool of so-called ribosome biogenesis factors includes diverse proteins with enzymatic and structural functions. Some of them have homologs in yeast S. cerevisiae, and their function can be inferred from the structural and biochemical data obtained for the yeast counterparts. The functions of human proteins RPF1 and ESF1 remain largely unclear, although RPF1 has been recently shown to participate in 60S biogenesis. Both proteins have drawn our attention since they contribute to the early stages of ribosome biogenesis, which are far less studied than the later stages. In this study, we employed the loss-of-function shRNA/siRNA-based approach to the human cell line HEK293 to determine the role of RPF1 and ESF1 in ribosome biogenesis. Downregulating RPF1 and ESF1 significantly changed the pattern of RNA products derived from 47S pre-rRNA. Our findings demonstrate that RPF1 and ESF1 are associated with different pre-ribosomal particles, pre-60S, and pre-40S particles, respectively. Our results allow for speculation about the particular steps of pre-rRNA processing, which highly rely on the RPF1 and ESF1 functions. We suggest that both factors are not directly involved in pre-rRNA cleavage but rather help pre-rRNA to acquire the conformation favoring its cleavage.
Assuntos
Precursores de RNA , Proteínas de Ligação a RNA , Humanos , Células HEK293 , Ribossomos/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.
Assuntos
COVID-19 , Ensaios de Triagem em Larga Escala , Biblioteca de Peptídeos , SARS-CoV-2 , Humanos , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/virologia , Ensaios de Triagem em Larga Escala/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Imunoglobulinas/imunologia , Imunoglobulinas/genética , Anticorpos Antivirais/imunologia , Mapeamento de Epitopos/métodosRESUMO
The biogenesis of ribosomes requires tightly controlled transcription and processing of pre-rRNA which comprises ribosomal RNAs forming the core of large and small ribosomal subunits. Early steps of the pre-rRNA processing and assembly of the ribosomal subunits require a large set of proteins that perform folding and nucleolytic cleavage of pre-rRNAs in the nucleoli. Structure and functions of proteins involved in the pre-rRNA processing have been extensively studied in the budding yeast S. cerevisiae. Functional characterization of their human homologues is complicated by the complexity of mammalian ribosomes and increased number of protein factors involved in the ribosomal biogenesis. Homologues of human nucleolar protein SURF6 from yeast and mouse, Rrp14 and Surf6, respectively, had been shown to be involved in the early steps of pre-rRNA processing. Rrp14 works as RNA chaperone in complex with proteins Ssf1 and Rrp15. Human SURF6 knockdown and overexpression were used to clarify a role of SURF6 in the early steps of pre-rRNA processing in human cell lines HeLa and HTC116. By analyzing the abundance of the rRNA precursors in cells with decreased level or overexpression of SURF6, we demonstrated that human SURF6 is involved in the maturation of rRNAs from both small and large ribosomal subunits. Changes in the SURF6 level caused by knockdown or overexpression of the protein do not result in the death of HeLa cells in contrast to murine embryonic fibroblasts, but significantly alter the distribution of cells among the phases of the cell cycle. SURF6 knockdown in both p53 sufficient and p53 deficient HCT116 human cancer cells results in elongation of G0/G1 and shortening of G2/M phase. This surprising result suggests p53 independence of SURF6 effects on the cell cycle and possible multiple functions of SURF6. Our data point to the shift from pathway 1 to pathway 2 of the rRNA biogenesis caused by the SURF6 knockdown and its likely association with p53 pathway.