Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.557
Filtrar
1.
BMC Genomics ; 22(1): 598, 2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362306

RESUMO

BACKGROUND: The typical single-chromosome mitochondrial (mt) genome of animals has fragmented into multiple minichromosomes in the lineage Mitodivisia, which contains most of the parasitic lice of eutherian mammals. These parasitic lice differ from each other even among congeneric species in mt karyotype, i.e. the number of minichromosomes, and the gene content and gene order in each minichromosome, which is in stark contrast to the extremely conserved single-chromosome mt genomes across most animal lineages. How fragmented mt genomes evolved is still poorly understood. We use Polyplax sucking lice as a model to investigate how tRNA gene translocation shapes the dynamic mt karyotypes. RESULTS: We sequenced the full mt genome of the Asian grey shrew louse, Polyplax reclinata. We then inferred the ancestral mt karyotype for Polyplax lice and compared it with the mt karyotypes of the three Polyplax species sequenced to date. We found that tRNA genes were entirely responsible for mt karyotype variation among these three species of Polyplax lice. Furthermore, tRNA gene translocation observed in Polyplax lice was only between different types of minichromosomes and towards the boundaries with the control region. A similar pattern of tRNA gene translocation can also been seen in other sucking lice with fragmented mt genomes. CONCLUSIONS: We conclude that inter-minichromosomal tRNA gene translocation orientated towards the boundaries with the control region is a major contributing factor to the highly dynamic mitochondrial genome organization in the parasitic lice of mammals.


Assuntos
Anoplura , Genoma Mitocondrial , Animais , Anoplura/genética , Genoma Mitocondrial/genética , Cariótipo , Mamíferos , Filogenia , RNA de Transferência/genética
2.
Nucleic Acids Res ; 49(15): 8757-8776, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379789

RESUMO

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tRNAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that the L-array of the model cyanobacterium Anabaena sp. PCC 7120, encoding 23 functional tRNAs, is largely induced upon impairment of the translation machinery. The cellular response to this challenge involves a global reprogramming of the transcriptome in two phases. tRNAs encoded in the array are induced in the second phase of the response, directly contributing to cell survival. Results presented here show that in some bacteria the tRNA gene set may be partitioned between a housekeeping subset, which constantly sustains translation, and an inducible subset that is generally silent but can provide functionality under particular conditions.


Assuntos
Genes Bacterianos , Óperon , Biossíntese de Proteínas , RNA de Transferência/genética , Estresse Fisiológico/genética , Anabaena/genética , Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Viabilidade Microbiana/genética , RNA de Transferência/metabolismo , Sequências Reguladoras de Ácido Nucleico
3.
Int J Mol Sci ; 22(16)2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34445460

RESUMO

Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status. Temperature sensitivity and protein aggregation are elevated in deg1 mutants and further increased in the presence of the ssd1-d allele, which encodes a truncated form of Ssd1. In addition, chronological lifespan is reduced in a deg1 ssd1-d mutant, and the negative genetic interactions of the U34 modifier genes ELP3 and URM1 with DEG1 are aggravated by ssd1-d. A loss of function mutation in SSD1, ELP3, and DEG1 induces pleiotropic and overlapping phenotypes, including sensitivity against target of rapamycin (TOR) inhibitor drug and cell wall stress by calcofluor white. Additivity in ssd1 deg1 double mutant phenotypes suggests independent roles of Ssd1 and tRNA modifications in TOR signaling and cell wall integrity. However, other tRNA modification defects cause growth and drug sensitivity phenotypes, which are not further intensified in tandem with ssd1-d. Thus, we observed a modification-specific rather than general effect of SSD1 status on phenotypic variation in tRNA modification mutants. Our results highlight how the cellular consequences of tRNA modification loss can be influenced by protein targeting specific mRNAs.


Assuntos
Transferases Intramoleculares/deficiência , Processamento Pós-Transcricional do RNA/genética , RNA Fúngico , RNA de Transferência , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Variação Biológica da População , Transferases Intramoleculares/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Mol Cell ; 81(16): 3243-3245, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34416137

RESUMO

Orellana et al. (2021) and Dai et al. (2021) demonstrate that increased m7G modification of a subset of tRNAs by the METTL1/WDR4 complex stabilizes these mRNAs against decay, increases translation efficiency, reduces ribosome pausing, is associated with poor survival in human cancers, and is directly transforming.


Assuntos
Neoplasias , Processamento Pós-Transcricional do RNA , Proteínas de Ligação ao GTP/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
5.
Mol Cell ; 81(16): 3339-3355.e8, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352206

RESUMO

Cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, but the underlying mechanisms are poorly understood. Here, we find that N7-methylguanosine (m7G) tRNA modification and its methyltransferase complex components, METTL1 and WDR4, are significantly upregulated in intrahepatic cholangiocarcinoma (ICC) and associated with poor prognosis. We further reveal the critical role of METTL1/WDR4 in promoting ICC cell survival and progression using loss- and gain-of-function assays in vitro and in vivo. Mechanistically, m7G tRNA modification selectively regulates the translation of oncogenic transcripts, including cell-cycle and epidermal growth factor receptor (EGFR) pathway genes, in m7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, using overexpression and knockout mouse models, we demonstrate the crucial oncogenic function of Mettl1-mediated m7G tRNA modification in promoting ICC tumorigenesis and progression in vivo. Our study uncovers the important physiological function and mechanism of METTL1-mediated m7G tRNA modification in the regulation of oncogenic mRNA translation and cancer progression.


Assuntos
Colangiocarcinoma/genética , Proteínas de Ligação ao GTP/genética , Metiltransferases/genética , Biossíntese de Proteínas , Animais , Carcinogênese/genética , Colangiocarcinoma/patologia , Progressão da Doença , Receptores ErbB/genética , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Camundongos , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética
6.
Mol Cell ; 81(16): 3323-3338.e14, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352207

RESUMO

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.


Assuntos
Carcinogênese/genética , Metiltransferases/genética , Neoplasias/genética , tRNA Metiltransferases/genética , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Metilação , Neoplasias/patologia , Oncogenes/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética
7.
Gene ; 802: 145868, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34364911

RESUMO

The Honduran white bat, Ectophylla alba (Allen 1982), is one of eight species belonging to the family Phyllostomidae that exclusively roosts in tents. Due to its restricted distribution, habitat specificity, and diet requirements, E. alba has been strongly affected by habitat loss and fragmentation during the last decade. In this study, we developed the first genomic resource for this species; we assembled and analyzed in detail the complete mitochondrial genome of E. alba. The mitogenome of E. alba is 16,664 bp in length and is comprised of 13 protein coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes (tRNAs), and a putative Control Region (CR) 1,232 bp in length. Gene arrangement in the mitochondrial chromosome of E. alba is identical to that reported before in other species of co-familiar bats. All PCGs are under purifying selection, with atp8 experiencing the least selective pressure. In all PCGs, codons ending with adenine are preferred over others ending in thymine and cytosine. Except tRNA-Serine 1, all tRNAs exhibit a cloverleaf secondary structure. The CR of E. alba exhibits three domains commonly described in other mammals, including bats; extended terminal associated sequences (ETAS), central, and conserved sequence block (CSB). A ML phylogenetic reconstruction of the family Phyllostomidae based on all 13 mitochondrial PCGs confirms the monophyletic status of the subfamily Sternodermatinae and indicates the close relationship between E. alba and the genus Artibeus. This is the first genomic resource developed for E. alba and represents the first step to improving our understanding of the genomic underpinnings involved in the evolution of specialization as well as acclimatization and adaptation to local and global change of specialist bats.


Assuntos
Quirópteros/genética , Genoma Mitocondrial , Animais , Quirópteros/classificação , Uso do Códon , DNA Mitocondrial , Filogenia , Proteínas/genética , RNA de Transferência/genética
8.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445114

RESUMO

The strong decoration of tRNAs with post-transcriptional modifications provides an unprecedented adaptability of this class of non-coding RNAs leading to the regulation of bacterial growth and pathogenicity. Accumulating data indicate that tRNA post-transcriptional modifications possess a central role in both the formation of bacterial cell wall and the modulation of transcription and translation fidelity, but also in the expression of virulence factors. Evolutionary conserved modifications in tRNA nucleosides ensure the proper folding and stability redounding to a totally functional molecule. However, environmental factors including stress conditions can cause various alterations in tRNA modifications, disturbing the pathogen homeostasis. Post-transcriptional modifications adjacent to the anticodon stem-loop, for instance, have been tightly linked to bacterial infectivity. Currently, advances in high throughput methodologies have facilitated the identification and functional investigation of such tRNA modifications offering a broader pool of putative alternative molecular targets and therapeutic avenues against bacterial infections. Herein, we focus on tRNA epitranscriptome shaping regarding modifications with a key role in bacterial infectivity including opportunistic pathogens of the human microbiome.


Assuntos
Bactérias/genética , Bactérias/patogenicidade , Transcriptoma/genética , Anticódon/genética , Humanos , Nucleosídeos/genética , Biossíntese de Proteínas/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Virulência/genética
9.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
10.
Nat Commun ; 12(1): 4913, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389722

RESUMO

Epitranscriptomic mechanisms linking tRNA function and the brain proteome to cognition and complex behaviors are not well described. Here, we report bi-directional changes in depression-related behaviors after genetic disruption of neuronal tRNA cytosine methylation, including conditional ablation and transgene-derived overexpression of Nsun2 in the mouse prefrontal cortex (PFC). Neuronal Nsun2-deficiency was associated with a decrease in tRNA m5C levels, resulting in deficits in expression of 70% of tRNAGly isodecoders. Altogether, 1488/5820 proteins changed upon neuronal Nsun2-deficiency, in conjunction with glycine codon-specific defects in translational efficiencies. Loss of Gly-rich proteins critical for glutamatergic neurotransmission was associated with impaired synaptic signaling at PFC pyramidal neurons and defective contextual fear memory. Changes in the neuronal translatome were also associated with a 146% increase in glycine biosynthesis. These findings highlight the methylation sensitivity of glycinergic tRNAs in the adult PFC. Furthermore, they link synaptic plasticity and complex behaviors to epitranscriptomic modifications of cognate tRNAs and the proteomic homeostasis associated with specific amino acids.


Assuntos
Transtorno Depressivo/fisiopatologia , Epigênese Genética/genética , Metiltransferases/genética , Proteoma/metabolismo , RNA de Transferência/genética , Transmissão Sináptica/genética , Animais , Transtorno Depressivo/genética , Transtorno Depressivo/metabolismo , Perfilação da Expressão Gênica/métodos , Metiltransferases/deficiência , Metiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/metabolismo , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiologia , Proteômica/métodos , RNA de Transferência/metabolismo , Transdução de Sinais/genética
11.
Nat Cell Biol ; 23(7): 684-691, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34253897

RESUMO

Members of the mammalian AlkB family are known to mediate nucleic acid demethylation1,2. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism3, but its function and mechanism of action are unknown. Here we report an approach to site-specifically detect N1-methyladenosine (m1A), N3-methylcytidine (m3C), N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m22G and m1A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA4-6. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.


Assuntos
Enzimas AlkB/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Enzimas AlkB/genética , Citidina/análogos & derivados , Citidina/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Biossíntese de Proteínas , RNA Mitocondrial/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo
12.
Methods Enzymol ; 656: 495-519, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34325796

RESUMO

With few exceptions, ribosomal protein synthesis begins with methionine (or its derivative N-formyl-methionine) across all domains of life. The role of methionine as the initiating amino acid is dictated by the unique structure of its cognate tRNA known as tRNAfMet. By mis-acylating tRNAfMet, we and others have shown that protein synthesis can be initiated with a variety of canonical and noncanonical amino acids both in vitro and in vivo. Furthermore, because the α-amine of the initiating amino acid is not required for peptide bond formation, translation can be initiated with a variety of structurally disparate carboxylic acids that bear little resemblance to traditional α-amino acids. Herein, we provide a detailed protocol to initiate in vitro protein synthesis with substituted benzoic acid and 1,3-dicarbonyl compounds. These moieties are introduced at the N-terminus of peptides by mis-acylated tRNAfMet, prepared by flexizyme-catalyzed tRNA acylation. In addition, we describe a protocol to initiate in vivo protein synthesis with aromatic noncanonical amino acids (ncAAs). This method relies on an engineered chimeric initiator tRNA that is acylated with ncAAs by an orthogonal aminoacyl-tRNA synthetase. Together, these systems are useful platforms for producing N-terminally modified proteins and for engineering the protein synthesis machinery of Escherichia coli to accept additional nonproteinogenic carboxylic acid monomers.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Escherichia coli/genética , Peptídeos , Proteínas , RNA de Transferência/genética
13.
Nat Commun ; 12(1): 4644, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330903

RESUMO

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Elongação Traducional da Cadeia Peptídica/genética , Fator G para Elongação de Peptídeos/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Biocatálise , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
14.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199774

RESUMO

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5'-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.


Assuntos
Mitocôndrias/enzimologia , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Ribonuclease P/metabolismo , Alelos , Animais , Drosophila melanogaster/genética , Mitocôndrias/patologia , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
15.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298922

RESUMO

The coordinated transcription of the genome is the fundamental mechanism in molecular biology. Transcription in eukaryotes is carried out by three main RNA polymerases: Pol I, II, and III. One basic problem is how a decrease in tRNA levels, by downregulating Pol III efficiency, influences the expression pattern of protein-coding genes. The purpose of this study was to determine the mRNA levels in the yeast mutant rpc128-1007 and its overdose suppressors, RBS1 and PRT1. The rpc128-1007 mutant prevents assembly of the Pol III complex and functionally mimics similar mutations in human Pol III, which cause hypomyelinating leukodystrophies. We applied RNAseq followed by the hierarchical clustering of our complete RNA-seq transcriptome and functional analysis of genes from the clusters. mRNA upregulation in rpc128-1007 cells was generally stronger than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the derepression of general transcription factor Gcn4, differently modulated by suppressor genes. rpc128-1007 mutation, regardless of the presence of suppressors, also resulted in a weak increase in the expression of ribosome biogenesis genes. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. In summary, our results provide the regulatory links affected by Pol III assembly that contribute differently to cellular fitness.


Assuntos
RNA Polimerase III/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação para Baixo/genética , Regulação Fúngica da Expressão Gênica/genética , Humanos , RNA Polimerase II/genética , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Transcriptoma/genética , Regulação para Cima/genética
16.
Methods Mol Biol ; 2277: 203-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080154

RESUMO

Here we summarize our latest efforts to elucidate the role of mtDNA variants affecting the mitochondrial translation machinery, namely variants mapping to the mt-rRNA and mt-tRNA genes. Evidence is accumulating to suggest that the cellular response to interference with mitochondrial translation is different from that occurring as a result of mutations in genes encoding OXPHOS proteins. As a result, it appears safe to state that a complete view of mitochondrial disease will not be obtained until we understand the effect of mt-rRNA and mt-tRNA variants on mitochondrial protein synthesis. Despite the identification of a large number of potentially pathogenic variants in the mitochondrially encoded rRNA (mt-rRNA) genes, we lack direct methods to firmly establish their pathogenicity. In the absence of such methods, we have devised an indirect approach named heterologous inferential analysis (HIA ) that can be used to make predictions concerning the disruptive potential of a large subset of mt-rRNA variants. We have used HIA to explore the mutational landscape of 12S and 16S mt-rRNA genes. Our HIA studies include a thorough classification of all rare variants reported in the literature as well as others obtained from studies performed in collaboration with physicians. HIA has also been used with non-mammalian mt-rRNA genes to elucidate how mitotypes influence the interaction of the individual and the environment. Regarding mt-tRNA variations, rapidly growing evidence shows that the spectrum of mutations causing mitochondrial disease might differ between the different mitochondrial haplogroups seen in human populations.


Assuntos
Biologia Computacional/métodos , DNA Mitocondrial/genética , Genômica/métodos , Doenças Mitocondriais/genética , RNA Mitocondrial/genética , Humanos , Mutação , Biossíntese de Proteínas , RNA Ribossômico , RNA Ribossômico 16S , RNA de Transferência/genética
17.
Genes (Basel) ; 12(6)2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071968

RESUMO

Cotton is one of the most important fiber and oil crops in the world. Chloroplast genomes harbor their own genetic materials and are considered to be highly conserved. Transfer RNAs (tRNAs) act as "bridges" in protein synthesis by carrying amino acids. Currently, the variation and evolutionary characteristics of tRNAs in the cotton chloroplast genome are poorly understood. Here, we analyzed the structural variation and evolution of chloroplast tRNA (cp tRNA) based on eight diploid and two allotetraploid cotton species. We also investigated the nucleotide evolution of chloroplast genomes in cotton species. We found that cp tRNAs in cotton encoded 36 or 37 tRNAs, and 28 or 29 anti-codon types with lengths ranging from 60 to 93 nucleotides. Cotton chloroplast tRNA sequences possessed specific conservation and, in particular, the Ψ-loop contained the conserved U-U-C-X3-U. The cp tRNAs of Gossypium L. contained introns, and cp tRNAIle contained the anti-codon (C-A-U), which was generally the anti-codon of tRNAMet. The transition and transversion analyses showed that cp tRNAs in cotton species were iso-acceptor specific and had undergone unequal rates of evolution. The intergenic region was more variable than coding regions, and non-synonymous mutations have been fixed in cotton cp genomes. On the other hand, phylogeny analyses indicated that cp tRNAs of cotton were derived from several inferred ancestors with greater gene duplications. This study provides new insights into the structural variation and evolution of chloroplast tRNAs in cotton plants. Our findings could contribute to understanding the detailed characteristics and evolutionary variation of the tRNA family.


Assuntos
Evolução Molecular , Genes de Cloroplastos , Variação Estrutural do Genoma , Gossypium/genética , RNA de Transferência/genética , Códon/genética , Gossypium/classificação , Filogenia
18.
Science ; 372(6546): 1057-1062, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34083482

RESUMO

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.


Assuntos
Códon , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/virologia , Compostos Macrocíclicos/metabolismo , Polímeros/metabolismo , Biossíntese de Proteínas , Fagos T/crescimento & desenvolvimento , Aminoácidos/metabolismo , Bacteriólise , Uso do Códon , Códon de Terminação , Evolução Molecular Direcionada , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Deleção de Genes , Código Genético , Genoma Bacteriano , Compostos Macrocíclicos/química , Mutagênese , Fatores de Terminação de Peptídeos/genética , Polímeros/química , RNA Bacteriano/genética , RNA de Transferência/genética , RNA de Transferência de Serina/genética , Ubiquitina/biossíntese , Ubiquitina/genética
19.
Methods Mol Biol ; 2323: 121-140, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086278

RESUMO

The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule-binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a profluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch-Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. In this updated chapter, we have added procedures on using biosensors in flow cytometry to detect exogenously added compounds. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.


Assuntos
Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Citometria de Fluxo/métodos , Corantes Fluorescentes/análise , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , RNA de Transferência/genética , RNA/genética , Riboswitch/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Compostos de Benzil , Clonagem Molecular/métodos , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imidazolinas , Isopropiltiogalactosídeo/farmacologia , Conformação de Ácido Nucleico , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Plasmídeos
20.
Methods Mol Biol ; 2323: 249-265, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086286

RESUMO

Research on RNA function and therapeutic potential is dominated by the use of chemoengineered RNA mimics. Recent efforts have led to the establishment of novel technologies for the production of recombinant or bioengineered RNA molecules, which should better recapitulate the structures, functions and safety profiles of natural RNAs because both are produced and folded in living cells. Herein, we describe a robust approach for reproducible fermentation production of bioengineered RNA agents (BERAs) carrying warhead miRNAs, siRNAs, aptamers, or other forms of small RNAs, based upon an optimal hybrid tRNA/pre-miRNA carrier. Target BERA/sRNAs are readily purified by fast protein liquid chromatography (FPLC) to a high degree of homogeneity (>97%). This approach offers a consistent high-level expression (>30% of total bacterial RNAs) and large-scale production of ready-to-use BERAs (multiple to tens milligrams from 1 L bacterial culture).


Assuntos
Bioengenharia/métodos , MicroRNAs/isolamento & purificação , RNA Bacteriano/isolamento & purificação , RNA de Transferência/isolamento & purificação , RNA não Traduzido/isolamento & purificação , RNA/isolamento & purificação , Sequência de Bases , Cromatografia por Troca Iônica/métodos , Clonagem Molecular/métodos , Contaminação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Endotoxinas/análise , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fermentação , MicroRNAs/biossíntese , MicroRNAs/genética , Desnaturação de Ácido Nucleico , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , RNA/biossíntese , RNA/genética , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , RNA de Transferência/biossíntese , RNA de Transferência/genética , RNA não Traduzido/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...