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1.
Gene ; 764: 145062, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32860900

RESUMO

Recently, DNA-based methods have proved to be accurate, fast and sensitive for meat authentication. According to the European Union, the food safety standards require accurate and detailed composition information of the meat products. Therefore, an accurate, fast and cost-effective identification methodology is needed. In this study, multiplex PCR coupled with 12S rDNA sequencing was employed for the detection of meat adulteration in two red meat products (frozen beef liver and cold cut samples, respectively) in Egypt. Multiplex PCR allowed the identification of ruminant, poultry, pork, and donkey residuals in processed red meat products (cold cuts) in a single step PCR reaction. Preliminary uniplex PCR was performed to evaluate primers specificity using DNA extracted from the positive control samples. The primers produced specific fragments for ruminant, poultry, pork, and donkey as follows: 271, 183, 531 and 145 bp, respectively. Multiplex PCR revealed that none of the samples was contaminated by porcine or donkey residuals, but 62.5% of all tested processed beef samples contained poultry contaminants. The sensitivity of this method was 0.01 ng/µL for beef, poultry and donkey and 0.1 ng/µL for pig. Another promising finding is the identification of all frozen beef liver samples as a cattle species (Bos taurus) through PCR-sequencing of a short fragment of 12S rRNA gene. Finally, we recommend the employment of multiplex PCR and PCR-sequencing of 12S rDNA for quality control in routine analysis of processed and frozen meat products.


Assuntos
Contaminação de Alimentos/análise , Indústria Alimentícia/normas , Produtos da Carne/análise , Reação em Cadeia da Polimerase Multiplex , RNA Ribossômico/genética , Animais , Bovinos/genética , Galinhas/genética , Egito , Limite de Detecção , Produtos da Carne/normas , Carne Vermelha/análise , Carne Vermelha/normas , Análise de Sequência de DNA/métodos , Especificidade da Espécie
2.
Mol Cell ; 79(6): 1024-1036.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32871103

RESUMO

Bacterial ribosomal RNAs are synthesized by a dedicated, conserved transcription-elongation complex that transcribes at high rates, shields RNA polymerase from premature termination, and supports co-transcriptional RNA folding, modification, processing, and ribosomal subunit assembly by presently unknown mechanisms. We have determined cryo-electron microscopy structures of complete Escherichia coli ribosomal RNA transcription elongation complexes, comprising RNA polymerase; DNA; RNA bearing an N-utilization-site-like anti-termination element; Nus factors A, B, E, and G; inositol mono-phosphatase SuhB; and ribosomal protein S4. Our structures and structure-informed functional analyses show that fast transcription and anti-termination involve suppression of NusA-stabilized pausing, enhancement of NusG-mediated anti-backtracking, sequestration of the NusG C-terminal domain from termination factor ρ, and the ρ blockade. Strikingly, the factors form a composite RNA chaperone around the RNA polymerase RNA-exit tunnel, which supports co-transcriptional RNA folding and annealing of distal RNA regions. Our work reveals a polymerase/chaperone machine required for biosynthesis of functional ribosomes.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Chaperonas Moleculares/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/ultraestrutura , Biossíntese de Proteínas/genética , Dobramento de RNA/genética , RNA Ribossômico/genética , RNA Ribossômico/ultraestrutura , Proteínas Ribossômicas/ultraestrutura , Ribossomos/ultraestrutura , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/ultraestrutura
3.
Nucleic Acids Res ; 48(18): 10397-10412, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946572

RESUMO

The RNA helicase RIG-I plays a key role in sensing pathogen-derived RNA. Double-stranded RNA structures bearing 5'-tri- or diphosphates are commonly referred to as activating RIG-I ligands. However, endogenous RNA fragments generated during viral infection via RNase L also activate RIG-I. Of note, RNase-digested RNA fragments bear a 5'-hydroxyl group and a 2',3'-cyclic phosphate. How endogenous RNA fragments activate RIG-I despite the lack of 5'-phosphorylation has not been elucidated. Here we describe an endogenous RIG-I ligand (eRL) that is derived from the internal transcribed spacer 2 region (ITS2) of the 45S ribosomal RNA after partial RNase A digestion in vitro, RNase A protein transfection or RNase L activation. The immunostimulatory property of the eRL is dependent on 2',3'-cyclic phosphate and its sequence is characterized by a G-quadruplex containing sequence motif mediating guanosine-5'-triphosphate (GTP) binding. In summary, RNase generated self-RNA fragments with 2',3'-cyclic phosphate function as nucleotide-5'-triphosphate binding aptamers activating RIG-I.


Assuntos
Proteína DEAD-box 58/genética , RNA Helicases/genética , RNA Ribossômico/genética , RNA/genética , Guanosina Trifosfato/genética , Humanos , Ligantes , Fosfatos/metabolismo , RNA/química , RNA Helicases/metabolismo , Ribonucleases/genética
4.
Gene ; 762: 145041, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32777523

RESUMO

Mitochondrial genome sequencing has become widely used in numerous fields, including systematics, phylogeny, and evolutionary genomics. To elucidate phylogenetic relationships among members of the family Characidae, we sequenced the mitogenomes of four species within this family, namely, Aphyocharax rathbuni, Hyphessobrycon herbertaxelrodi, Hyphessobrycon megalopterus, and Prionobrama filigera. The mitogenomes were found to be 16,678-16,841 bp and encode 37 typical mitochondrial genes (13 protein-coding, 2 ribosomal RNA, and 22 transfer RNA genes). Gene arrangements in the studied species are consistent with those in the inferred ancestral fish. Most protein-coding genes in these mitogenomes have typical ATN start codons and TAR or an incomplete stop codon T-. Phylogenetic relationships based on Bayesian inference and maximum-likelihood methods indicated that A. rathbuni, H. herbertaxelrodi, H. megalopterus, and P. filigera belong to the Characidae family. Of the 15 Characidae species studied, three pairs were of the same genus, but the results for only one pair were well supported. This phylogenetic classification is inconsistent with those described in previous morphological and taxonomic studies on this family. Thus, systematic classification of the Characidae requires further examination. Our findings yield new mitogenomic data that will provide a basis for future phylogenetic and taxonomic studies.


Assuntos
Caraciformes/genética , Genoma Mitocondrial , Filogenia , Animais , Caraciformes/classificação , Códon/genética , Anotação de Sequência Molecular , Fases de Leitura Aberta , RNA Ribossômico/genética , RNA de Transferência/genética
5.
BMC Evol Biol ; 20(1): 88, 2020 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-32682389

RESUMO

BACKGROUND: Old World porcupines (Family: Hystricidae) are the third-largest rodents and inhabit southern Europe, Asia, and most regions of Africa. They are a typical indicator of warm climate and their distribution is restricted to tropical and subtropical zones. In China, porcupines are widely distributed in southern areas of the Yangtze River. However, fossil remains have been identified in a few sites in northern China, among which Tianyuan Cave-near Zhoukoudian site-represents the latest known porcupine fossil record. So far, studies have focused mainly on porcupines' husbandry and domestication but little is known about their intrafamilial phylogenetic relationships and evolutionary history. RESULTS: In this study, we sequence partial mitochondrial 12S rRNA and cyt b genes for seven Late Pleistocene porcupine individuals from Northern, Southern and Central China. Phylogenetic analyses show that the Tianyuan Cave porcupines, which had been morphologically identified as Hystrix subcristata, have a closer relationship to Hystrix brachyura. CONCLUSION: Together with morphological adaptation characteristics, associated fauna, and climate change evidence, the molecular results reveal that a Late Quaternary extirpation has occurred during the evolutionary history of porcupines.


Assuntos
Evolução Biológica , DNA Antigo , Fósseis , Porcos-Espinhos/anatomia & histologia , Porcos-Espinhos/genética , Animais , China , Geografia , Funções Verossimilhança , Filogenia , RNA Ribossômico/genética , Especificidade da Espécie , Fatores de Tempo
6.
Parasitol Res ; 119(8): 2733-2740, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32617726

RESUMO

Amebiasis is a worldwide parasitic zoonosis, with symptoms of abdominal discomfort, indigestion, diarrhea, and even death. However, limited information about the prevalence of Entamoeba spp. in experimental nonhuman primates (NHPs) in southwestern China is available. The objective of the current study was to investigate the frequency and species identity of Entamoeba to evaluate potential zoonotic risk factors for Entamoeba spp. infection in experimental NHPs. A total of 505 fecal samples were collected from NHPs (macaques) and analyzed by PCR analysis the small subunit rRNA (SSU rRNA) gene of Entamoeba spp. Forty-seven specimens were positive for Entamoeba spp., and the prevalence of Entamoeba spp. was 9.31% (47/505). Significant differences in the prevalence rates among the three breeds (P = 0.002 < 0.01, df = 2, χ2 = 12.33) and feed types (P = 0.001 < 0.01, df = 1, χ2 = 10.12) were observed. Altogether, four Entamoeba species, including E. dispar (57.44%), E. chattoni (29.78%), E. histolytica (6.38%), and E. coli (6.38%), were identified by DNA sequence analysis. The results suggested a low prevalence but high diversity of Entamoeba species in experimental NHPs in Yunnan Province, southwestern China. Results of this study contribute to the knowledge of the genetic characteristics of Entamoeba spp. in NHPs.


Assuntos
Entamoeba/genética , Entamebíase/veterinária , Macaca/parasitologia , Infecções Protozoárias em Animais/epidemiologia , Infecções Protozoárias em Animais/parasitologia , Animais , Animais de Laboratório , China/epidemiologia , DNA de Protozoário/genética , Entamoeba/classificação , Entamoeba/isolamento & purificação , Entamebíase/epidemiologia , Entamebíase/parasitologia , Entamebíase/transmissão , Fezes/parasitologia , Epidemiologia Molecular , Prevalência , Infecções Protozoárias em Animais/transmissão , RNA Ribossômico/genética , Subunidades Ribossômicas Menores/genética , Análise de Sequência de DNA
7.
Nucleic Acids Res ; 48(15): 8360-8373, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32619236

RESUMO

Coordinated regulation of ribosomal RNA (rRNA) synthesis and ribosomal protein gene (RPG) transcription by eukaryotic RNA polymerases (RNAP) is a key requirement for growth control. Although evidence for balance between RNPI-dependent 35S rRNA production and RNAPII-mediated RPG transcription have been described, the molecular basis is still obscure. Here, we found that Rph1 modulates the transcription status of both rRNAs and RPGs in yeast. We show that Rph1 widely associates with RNAPI and RNAPII-transcribed genes. Deletion of RPH1 remarkably alleviates cell slow growth caused by TORC1 inhibition via derepression of rRNA and RPG transcription under nutrient stress conditions. Mechanistically, Rim15 kinase phosphorylates Rph1 upon rapamycin treatment. Phosphorylation-mimetic mutant of Rph1 exhibited more resistance to rapamycin treatment, decreased association with ribosome-related genes, and faster cell growth compared to the wild-type, indicating that Rph1 dissociation from chromatin ensures cell survival upon nutrient stress. Our results uncover the role of Rph1 in coordination of RNA polymerases-mediated transcription to control cell growth under nutrient stress conditions.


Assuntos
Proliferação de Células/genética , Histona Desmetilases/genética , Proteínas Quinases/genética , RNA Ribossômico/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Cromatina/genética , Regulação Fúngica da Expressão Gênica/genética , Fosforilação , Proteínas Ribossômicas/genética , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Transcrição Genética
8.
PLoS Genet ; 16(6): e1008511, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32598339

RESUMO

Ribosomal DNA (rDNA) is the most transcribed genomic region and contains hundreds of tandem repeats. Maintaining these rDNA repeats as well as the level of rDNA transcription is essential for cellular homeostasis. DNA damages generated in rDNA need to be efficiently and accurately repaired and rDNA repeats instability has been reported in cancer, aging and neurological diseases. Here, we describe that the histone demethylase JMJD6 is rapidly recruited to nucleolar DNA damage and is crucial for the relocalisation of rDNA in nucleolar caps. Yet, JMJD6 is dispensable for rDNA transcription inhibition. Mass spectrometry analysis revealed that JMJD6 interacts with the nucleolar protein Treacle and modulates its interaction with NBS1. Moreover, cells deficient for JMJD6 show increased sensitivity to nucleolar DNA damage as well as loss and rearrangements of rDNA repeats upon irradiation. Altogether our data reveal that rDNA transcription inhibition is uncoupled from rDNA relocalisation into nucleolar caps and that JMJD6 is required for rDNA stability through its role in nucleolar caps formation.


Assuntos
Dano ao DNA , Histona Desmetilases com o Domínio Jumonji/genética , RNA Ribossômico/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , RNA Ribossômico/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(26): 15137-15147, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554502

RESUMO

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations in DKC1, NOP10, or NHP2 cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition. Here, we report a phenotype comprising nephrotic syndrome, cataracts, sensorineural deafness, enterocolitis, and early lethality in two pedigrees: males with DKC1 p.Glu206Lys and two children with homozygous NOP10 p.Thr16Met. Females with heterozygous DKC1 p.Glu206Lys developed cataracts and sensorineural deafness, but nephrotic syndrome in only one case of skewed X-inactivation. We found telomere attrition in both pedigrees, but no mucocutaneous abnormalities suggestive of DC. Both mutations fall at the dyskerin-NOP10 binding interface in a region distinct from those implicated in DC, impair the dyskerin-NOP10 interaction, and disrupt the catalytic pseudouridylation site. Accordingly, we found reduced pseudouridine levels in the ribosomal RNA (rRNA) of the patients. Zebrafish dkc1 mutants recapitulate the human phenotype and show reduced 18S pseudouridylation, ribosomal dysregulation, and a cell-cycle defect in the absence of telomere attrition. We therefore propose that this human disorder is the consequence of defective snoRNP pseudouridylation and ribosomal dysfunction.


Assuntos
Catarata/genética , Proteínas de Ciclo Celular/genética , Enterocolite/genética , Perda Auditiva Neurossensorial/genética , Síndrome Nefrótica/genética , Proteínas Nucleares/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Animais , Criança , Feminino , Predisposição Genética para Doença , Humanos , Longevidade , Masculino , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Linhagem , Conformação Proteica , RNA Ribossômico/genética , Peixe-Zebra
10.
Parasitol Res ; 119(8): 2741-2745, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32577820

RESUMO

Blastocystis is a zoonotic protozoan parasite frequently identified in the intestinal tract of humans and a vast variety of animals, worldwide. Here, we assessed the prevalence of Blastocystis and its subtypes in stool samples of raccoons. Stool samples from 30 raccoons were collected. Total DNA was extracted, and the barcoding region of the small subunit ribosomal rRNA (SSU rRNA) gene was amplified and sequenced. Specific fragment for Blastocystis was successfully amplified in five samples (16.66%). Sequencing analysis revealed ST1, ST2, and ST3 among 1, 2, and 2 Blastocystis-positive samples. Our results documented the presence of Blastocystis subtypes 1-3 in raccoons. Subtype 1 showed higher similarity to the human isolates of Blastocystis. However, it seems that raccoons may emerge as reservoirs for Blastocystis and may be linked to zoonotic transmission of the protist.


Assuntos
Infecções por Blastocystis/veterinária , Blastocystis/isolamento & purificação , Infecções Protozoárias em Animais/parasitologia , Guaxinins/parasitologia , Animais , Sequência de Bases , Blastocystis/classificação , Blastocystis/genética , Infecções por Blastocystis/epidemiologia , Infecções por Blastocystis/parasitologia , Infecções por Blastocystis/transmissão , DNA de Protozoário/genética , Fezes/parasitologia , Variação Genética , Genótipo , Irã (Geográfico)/epidemiologia , Prevalência , Infecções Protozoárias em Animais/epidemiologia , Infecções Protozoárias em Animais/transmissão , RNA Ribossômico/genética , Subunidades Ribossômicas Menores/genética
11.
RNA ; 26(10): 1481-1488, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32503920

RESUMO

Ribosome footprint profiling is a high-throughput sequencing-based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally very abundant, ribosomal RNA sequences that dominate libraries and increase sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued as a standalone product and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them generally unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.


Assuntos
Biossíntese de Proteínas/genética , Ribonucleases/genética , Ribossomos/genética , Animais , Viés , Linhagem Celular , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Células K562 , Mamíferos , Camundongos , RNA/genética , RNA Ribossômico/genética , Ratos , Análise de Sequência de RNA/métodos
12.
RNA ; 26(10): 1360-1379, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32503921

RESUMO

Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their respective activities. This work describes how the yeast prefoldin-like Bud27 protein, which physically interacts with the Rpb5 common subunit of the three RNA polymerases, is able to modulate the transcription mediated by the RNA polymerase I, likely by influencing transcription elongation, the transcription of the RNA polymerase III, and the processing of ribosomal RNA. Bud27 also regulates both RNA polymerase II-dependent transcription of ribosomal proteins and ribosome biogenesis regulon genes, likely by occupying their DNA ORFs, and the processing of the corresponding mRNAs. With RNA polymerase II, this association occurs in a transcription rate-dependent manner. Our data also indicate that Bud27 inactivation alters the phosphorylation kinetics of ribosomal protein S6, a readout of TORC1 activity. We conclude that Bud27 impacts the homeostasis of the ribosome biogenesis process by regulating the activity of the three RNA polymerases and, in this way, the synthesis of ribosomal components. This quite likely occurs through a functional connection of Bud27 with the TOR signaling pathway.


Assuntos
Chaperonas Moleculares/genética , Fatores de Iniciação de Peptídeos/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Genética/genética , Núcleo Celular/genética , RNA Polimerase II/genética , RNA Polimerase III/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/genética
13.
Mol Phylogenet Evol ; 150: 106861, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32497832

RESUMO

Southeast Asia hosts a rich concentration of biodiversity within multiple biodiversity hotspots. Indochina, a region with remarkably high levels of in situ diversification, possesses five major rivers (Ayeyarwady, Chiang Mai, Mekong, Red, and Salween), several of which coincide with phylogenetic breaks of terrestrial taxa. Draco maculatus possesses a range that stretches across Indochina, which widespread geographic distribution along with potential discrete variation within subspecies alludes to the possibility of this taxon constituting multiple divergent lineages. Using sequence data from three mitochondrial (12S, 16S, and ND2) and three nuclear (BDNF, CMOS, and PNN) genes, we provide the first estimated phylogeny of this hypothesized species complex and examine its phylogeographic architecture with maximum likelihood and Bayes factor delimitation (BFD) approaches. Our results support multiple divergent lineages with phylogenetic breaks coincident with rivers, indicating that river barriers may be contributing to the elevated levels of in situ diversification of Indochina.


Assuntos
Lagartos/classificação , Animais , Teorema de Bayes , Biodiversidade , Fator Neurotrófico Derivado do Encéfalo/classificação , Fator Neurotrófico Derivado do Encéfalo/genética , Indochina , Lagartos/genética , Mitocôndrias/genética , NADH Desidrogenase/classificação , NADH Desidrogenase/genética , Filogenia , Filogeografia , Subunidades Proteicas/classificação , Subunidades Proteicas/genética , RNA Ribossômico/classificação , RNA Ribossômico/genética
14.
Int J Syst Evol Microbiol ; 70(5): 3449-3454, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32375951

RESUMO

Plants are important reservoirs of described and undescribed species of yeast. During a study of yeasts associated with bromeliads from the Northeast region of Brazil (collected in 2013-2017), analysis of the D1/D2 domain of the LSU rRNA and internal transcribed spacer (ITS) region identified eleven strains of yeasts as representing an unknown species of the genus Vishniacozyma. The species may have a diverse habitat in Brazil as a strain was collected from a flowering plant (Acanthaceae) in 1994. As a consequence, we propose Vishniacozyma alagoana sp. nov. as a member of the tremellomycetes yeasts (Agaricomycotina, Basidiomycota). Vishniacozyma alagoana sp. nov. was found in Atlantic Forest (a tropical rainforest) and the Caatinga (a seasonally dry tropical forest) associated with bromeliads in northeast and southeastern Brazil. The proposed novel species is related to Vishniacozyma taibaiensis and distinguished by eight nucleotide substitutions in the D1/D2 domain and seventeen in the ITS region. In addition, Vishniacozyma alagoana sp. nov. differs from V. taibaiensis by the ability to assimilate ribitol. The holotype is CBS 15966T.


Assuntos
Basidiomycota/classificação , Bromeliaceae/microbiologia , Filogenia , Floresta Úmida , Basidiomycota/isolamento & purificação , Brasil , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Técnicas de Tipagem Micológica , RNA Ribossômico/genética , Análise de Sequência de DNA
15.
RNA ; 26(9): 1268-1282, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32467310

RESUMO

PPR proteins are a diverse family of RNA binding factors found in all Eukaryotic lineages. They perform multiple functions in the expression of organellar genes, mostly on the post-transcriptional level. PPR proteins are also significant determinants of evolutionary nucleo-organellar compatibility. Plant PPR proteins recognize their RNA substrates using a simple modular code. No target sequences recognized by animal or yeast PPR proteins were identified prior to the present study, making it impossible to assess whether this plant PPR code is conserved in other organisms. Dmr1p (Ccm1p, Ygr150cp) is a S. cerevisiae PPR protein essential for mitochondrial gene expression and involved in the stability of 15S ribosomal RNA. We demonstrate that in vitro Dmr1p specifically binds a motif composed of multiple AUA repeats occurring twice in the 15S rRNA sequence as the minimal 14 nt (AUA)4AU or longer (AUA)7 variant. Short RNA fragments containing this motif are protected by Dmr1p from exoribonucleolytic activity in vitro. Presence of the identified motif in mtDNA of different yeast species correlates with the compatibility between their Dmr1p orthologs and S. cerevisiae mtDNA. RNA recognition by Dmr1p is likely based on a rudimentary form of a PPR code specifying U at every third position, and depends on other factors, like RNA structure.


Assuntos
Proteínas Mitocondriais/genética , Motivos de Nucleotídeos/genética , RNA Ribossômico/genética , RNA/genética , Subunidades Ribossômicas Menores/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Expressão Gênica/genética , Mitocôndrias/genética , Ribossomos/genética
16.
RNA ; 26(8): 1049-1059, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32371454

RESUMO

Nop9 is an essential factor in the processing of preribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its target RNA within the 20S preribosomal RNA. This structure reveals that Nop9 brings together a carboxy-terminal module recognizing the 5' single-stranded region of the RNA and a bifunctional amino-terminal module recognizing the central double-stranded stem region. We further show that the 3' single-stranded region of the 20S target RNA adds sequence-independent binding energy to the RNA-Nop9 interaction. Both the amino- and carboxy-terminal modules retain the characteristic sequence-specific recognition of PUF proteins, but the amino-terminal module has also evolved a distinct interface, which allows Nop9 to recognize either single-stranded RNA sequences or RNAs with a combination of single-stranded and structured elements.


Assuntos
RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X/métodos , Humanos , Conformação Proteica em alfa-Hélice/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
17.
PLoS One ; 15(4): e0225233, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324729

RESUMO

The Assam Roofed Turtle, Pangshura sylhetensis is an endangered and least studied species endemic to India and Bangladesh. The present study decodes the first complete mitochondrial genome of P. sylhetensis (16,568 bp) by using next-generation sequencing. The assembly encodes 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and one control region (CR). Most of the genes were encoded on the majority strand, except NADH dehydrogenase subunit 6 (nad6) and eight tRNAs. All PCGs start with an ATG initiation codon, except for Cytochrome oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 5 (nad5), which both start with GTG codon. The study also found the typical cloverleaf secondary structures in most of the predicted tRNA structures, except for serine (trnS1) which lacks of conventional DHU arm and loop. Both Bayesian and maximum-likelihood phylogenetic inference using 13 concatenated PCGs demonstrated strong support for the monophyly of all 52 Testudines species within their respective families and revealed Batagur trivittata as the nearest neighbor of P. sylhetensis. The mitogenomic phylogeny with other amniotes is congruent with previous research, supporting the sister relationship of Testudines and Archosaurians (birds and crocodilians). Additionally, the mitochondrial Gene Order (GO) analysis indicated plesiomorphy with the typical vertebrate GO in most of the Testudines species.


Assuntos
Espécies em Perigo de Extinção , Genoma Mitocondrial , Tartarugas/genética , Animais , Ordem dos Genes , Filogenia , RNA Ribossômico/genética , RNA de Transferência/genética , Proteínas de Répteis/genética
18.
Nat Commun ; 11(1): 1858, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313034

RESUMO

Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.


Assuntos
Bactérias/metabolismo , Engenharia de Proteínas/métodos , Ribossomos/química , Biologia Sintética/métodos , Alelos , Sistema Livre de Células , Cristalografia por Raios X , Modelos Moleculares , Modelos Teóricos , Conformação Molecular , Mutação , Peptídeos/química , Peptidil Transferases/química , Plasmídeos/genética , Biossíntese de Proteínas , Proteoma , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico 23S/genética , Thermus thermophilus/química
19.
Mol Cell ; 78(4): 670-682.e8, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32343944

RESUMO

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.


Assuntos
Caulobacter crescentus/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Organelas/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/crescimento & desenvolvimento , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Humanos , Complexos Multienzimáticos/genética , Organelas/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/genética , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
20.
RNA ; 26(8): 1069-1078, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32345633

RESUMO

A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative nonribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (depletion of abundant sequences by hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH uses the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56%-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron DASH works robustly, even with subnanogram amounts of input RNA. Its efficiency, high sensitivity, ease of implementation, and low cost (∼$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.


Assuntos
Sistemas CRISPR-Cas/genética , RNA Bacteriano/genética , RNA Ribossômico/genética , RNA-Seq/métodos , Bactérias/genética , DNA Complementar/genética , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hibridização de Ácido Nucleico/métodos , Análise de Sequência de RNA/métodos
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