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1.
Gene ; 745: 144640, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32247037

RESUMO

Codon usage bias is an important genomic phenomenon, where highly expressed genes use optimal codons for smoother translation with high yield, facilitated by the cognate tRNAs. Here, we presented the tRNA co-adaptation index (co-AI) by correlating tRNA gene copy number and codon composition in Saccharomyces cerevisiae. We observed that this co-AI is positively correlated with protein abundance and translation rate. Considering nucleotide substitutions, co-AI influences synonymous substitutions more than gene expression and protein abundance, the most important determinants of evolutionary rate. Co-AI correlates positively with mRNA secondary structure stability and mRNA half-life, which may lead to protein accumulation under high co-AI. However, the highly expressed proteins encoded by high co-AI genes are assisted by molecular chaperones to attain their proper functional conformation and prevent accumulation.


Assuntos
Dosagem de Genes , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Mutação Silenciosa , Uso do Códon , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
2.
Nature ; 580(7803): 396-401, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296180

RESUMO

Cancer genomics has revealed many genes and core molecular processes that contribute to human malignancies, but the genetic and molecular bases of many rare cancers remains unclear. Genetic predisposition accounts for 5 to 10% of cancer diagnoses in children1,2, and genetic events that cooperate with known somatic driver events are poorly understood. Pathogenic germline variants in established cancer predisposition genes have been recently identified in 5% of patients with the malignant brain tumour medulloblastoma3. Here, by analysing all protein-coding genes, we identify and replicate rare germline loss-of-function variants across ELP1 in 14% of paediatric patients with the medulloblastoma subgroup Sonic Hedgehog (MBSHH). ELP1 was the most common medulloblastoma predisposition gene and increased the prevalence of genetic predisposition to 40% among paediatric patients with MBSHH. Parent-offspring and pedigree analyses identified two families with a history of paediatric medulloblastoma. ELP1-associated medulloblastomas were restricted to the molecular SHHα subtype4 and characterized by universal biallelic inactivation of ELP1 owing to somatic loss of chromosome arm 9q. Most ELP1-associated medulloblastomas also exhibited somatic alterations in PTCH1, which suggests that germline ELP1 loss-of-function variants predispose individuals to tumour development in combination with constitutive activation of SHH signalling. ELP1 is the largest subunit of the evolutionarily conserved Elongator complex, which catalyses translational elongation through tRNA modifications at the wobble (U34) position5,6. Tumours from patients with ELP1-associated MBSHH were characterized by a destabilized Elongator complex, loss of Elongator-dependent tRNA modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response, consistent with loss of protein homeostasis due to Elongator deficiency in model systems7-9. Thus, genetic predisposition to proteome instability may be a determinant in the pathogenesis of paediatric brain cancers. These results support investigation of the role of protein homeostasis in other cancer types and potential for therapeutic interference.


Assuntos
Neoplasias Cerebelares/metabolismo , Mutação em Linhagem Germinativa , Meduloblastoma/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Criança , Feminino , Humanos , Masculino , Meduloblastoma/genética , Linhagem , RNA de Transferência/metabolismo , Fatores de Elongação da Transcrição/genética
3.
Mol Biol (Mosk) ; 54(1): 78-86, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32163391

RESUMO

Short Interspersed Elements (SINEs) are mobile genetic elements of higher eukaryotes, which originated during evolution from various tRNAs and less often from 5S rRNA and 7SL RNA. Similar to the genes of these RNAs, SINEs are transcribed by RNA polymerase III. The transcripts of some mammalian SINEs have an ability to undergo AAUAAA-dependent polyadenylation, which is unique for the RNA polymerase III transcripts. It is well known that this polyadenylation of many RNA polymerase II transcripts (e.g., mRNAs) increases their lifetime in the cell. The aim of this work is to examine whether the stability of SINE transcripts increases as a result of AAUAAA-dependent polyadenylation. HeLa cells were transfected with SINE DNA, both containing and not containing the polyadenylation signal (AATAAA). One day later, the transcription was inhibited by actinomycin D, and the decrease in the level of the SINE transcripts was monitored by northern hybridization. For all the eight studied SINEs, the half-life of nonpolyadenylated transcripts was 20-30 minutes, and for polyadenylated transcripts, this parameter exceeded 3 hours. Interestingly, the insertion of an additional 80-bp DNA fragment into the middle region of B2 SINE did not significantly reduce the stability of the polyadenylated transcripts. It is most likely that the increase in the lifetime of the polyadenylated SINE transcripts is due to the fact that the poly(A) tail interacts with the poly(A)-binding proteins (PABPs), thus protecting the RNA from degradation by the exonucleases acting from the 3'-end. The results make it possible to design SINE-based vectors intended for the expression of short noncoding RNAs, which are stable in a cell due to polyadenylation.


Assuntos
Poliadenilação , RNA Polimerase III/metabolismo , Estabilidade de RNA , RNA de Transferência/química , RNA de Transferência/metabolismo , Elementos Nucleotídeos Curtos e Dispersos/genética , Transcrição Genética , Animais , Células HeLa , Humanos , RNA de Transferência/genética
4.
PLoS One ; 15(2): e0229103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053677

RESUMO

Chemical modifications that regulate protein expression at the translational level are emerging as vital components of the cellular stress response. Transfer RNAs (tRNAs) are significant targets for methyl-based modifications, which are catalyzed by tRNA methyltransferases (Trms). Here, Saccharomyces cerevisiae served as a model eukaryote system to investigate the role of 2'-O-ribose tRNA methylation in the cell's response to oxidative stress. Using 2'-O-ribose deletion mutants for trms 3, 7, 13, and 44, in acute and chronic exposure settings, we demonstrate a broad cell sensitivity to oxidative stress-inducing toxicants (i.e., hydrogen peroxide, rotenone, and acetic acid). A global analysis of hydrogen peroxide-induced tRNA modifications shows a complex profile of decreased, or undetectable, 2'-O-ribose modification events in 2'-O-ribose trm mutant strains, providing a critical link between this type of modification event and Trm status post-exposure. Based on the pronounced oxidative stress sensitivity observed for trm7 mutants, we used a bioinformatic tool to identify transcripts as candidates for regulation by Trm7-catalyzed modifications (i.e., enriched in UUC codons decoded by tRNAPheGmAA). This screen identified transcripts linked to diverse biological processes that promote cellular recovery after oxidative stress exposure, including DNA repair, chromatin remodeling, and nutrient acquisition (i.e., CRT10, HIR3, HXT2, and GNP1); moreover, these mutants were also oxidative stress-sensitive. Together, these results solidify a role for TRM3, 7, 13, and 44, in the cellular response to oxidative stress, and implicate 2'-O-ribose tRNA modification as an epitranscriptomic strategy for oxidative stress recovery.


Assuntos
RNA de Transferência/metabolismo , Ribose/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Reparo do DNA/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
5.
PLoS One ; 15(2): e0228730, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053646

RESUMO

Messenger RNA (mRNA) degradation plays a critical role in regulating transcript levels in the cell and is a major control point for modulating gene expression. In yeast and other model organisms, codon identity is a powerful determinant of transcript stability, contributing broadly to impact half-lives. General principles governing mRNA stability are poorly understood in mammalian systems. Importantly, however, the degradation machinery is highly conserved, thus it seems logical that mammalian transcript half-lives would also be strongly influenced by coding determinants. Herein we characterize the contribution of coding sequence towards mRNA decay in human and Chinese Hamster Ovary cells. In agreement with previous studies, we observed that synonymous codon usage impacts mRNA stability in mammalian cells. Surprisingly, however, we also observe that the amino acid content of a gene is an additional determinant correlating with transcript stability. The impact of codon and amino acid identity on mRNA decay appears to be associated with underlying tRNA and intracellular amino acid concentrations. Accordingly, genes of similar physiological function appear to coordinate their mRNA stabilities in part through codon and amino acid content. Together, these results raise the possibility that intracellular tRNA and amino acid levels interplay to mediate coupling between translational elongation and mRNA degradation rate in mammals.


Assuntos
Aminoácidos/metabolismo , RNA Mensageiro/metabolismo , Animais , Células CHO , Códon , Cricetinae , Cricetulus , Meia-Vida , Células HeLa , Humanos , Fases de Leitura Aberta , Estabilidade de RNA , RNA de Transferência/metabolismo
6.
Nat Commun ; 11(1): 926, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066737

RESUMO

The field of epitranscriptomics continues to reveal how post-transcriptional modification of RNA affects a wide variety of biological phenomena. A pivotal challenge in this area is the identification of modified RNA residues within their sequence contexts. Mass spectrometry (MS) offers a comprehensive solution by using analogous approaches to shotgun proteomics. However, software support for the analysis of RNA MS data is inadequate at present and does not allow high-throughput processing. Existing software solutions lack the raw performance and statistical grounding to efficiently handle the numerous modifications found on RNA. We present a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), that addresses these shortcomings. We demonstrate the capability of NASE to reliably identify a wide range of modified RNA sequences in four original datasets of varying complexity. In human tRNA, we characterize over 20 different modification types simultaneously and find many cases of incomplete modification.


Assuntos
Epigenômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Processamento Pós-Transcricional do RNA/genética , Ferramenta de Busca , Espectrometria de Massas em Tandem/métodos , Sequência de Bases/genética , Bases de Dados Factuais/estatística & dados numéricos , Conjuntos de Dados como Assunto , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Reprodutibilidade dos Testes
7.
Nucleic Acids Res ; 48(6): e32, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-31974573

RESUMO

In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. Here, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and transport of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA mobility in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of long-term potentiation (LTP) in culture revealed up-regulation of mRNA translation with a similar effect in dendrites and somata, which appeared to be GluR-dependent 6 h post-activation. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Neurônios/metabolismo , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Animais , Compartimento Celular , Células Cultivadas , Dendritos/metabolismo , Corantes Fluorescentes/metabolismo , Potenciação de Longa Duração , Masculino , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
8.
Nucleic Acids Res ; 48(5): 2271-2286, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980822

RESUMO

The study of RNA expression is the fastest growing area of genomic research. However, despite the dramatic increase in the number of sequenced transcriptomes, we still do not have accurate estimates of the number and expression levels of non-coding RNA genes. Non-coding transcripts are often overlooked due to incomplete genome annotation. In this study, we use annotation-independent detection of RNA reads generated using a reverse transcriptase with low structure bias to identify non-coding RNA. Transcripts between 20 and 500 nucleotides were filtered and crosschecked with non-coding RNA annotations revealing 111 non-annotated non-coding RNAs expressed in different cell lines and tissues. Inspecting the sequence and structural features of these transcripts indicated that 60% of these transcripts correspond to new snoRNA and tRNA-like genes. The identified genes exhibited features of their respective families in terms of structure, expression, conservation and response to depletion of interacting proteins. Together, our data reveal a new group of RNA that are difficult to detect using standard gene prediction and RNA sequencing techniques, suggesting that reliance on actual gene annotation and sequencing techniques distorts the perceived architecture of the human transcriptome.


Assuntos
Anotação de Sequência Molecular/métodos , RNA Mensageiro/genética , RNA Nucleolar Pequeno/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Transcriptoma , Animais , Pareamento de Bases , Sequência de Bases , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Filogenia , RNA Mensageiro/classificação , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/classificação , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/classificação , RNA de Transferência/metabolismo , RNA não Traduzido/classificação , RNA não Traduzido/metabolismo , Análise de Sequência de RNA , Sequenciamento Completo do Exoma
9.
Biochim Biophys Acta Gene Regul Mech ; 1863(3): 194490, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31991215

RESUMO

The eukaryotic ribosomal protein uS19 has a C-terminal tail that is absent in its bacterial homologue. This tail has been shown to be involved in the formation of the decoding site of human ribosomes. We studied here the previously unexplored functional significance of the 15 C-terminal amino acid residues of human uS19 for the assembly of ribosomes and translation using HEK293-based cell cultures capable of producing FLAG-labeled uS19 (uS19FLAG) or its mutant form deprived of the mentioned amino acid ones. The examination of polysome profiles of cytoplasmic extracts from the respective cells revealed that the deletion of the above uS19 amino acid residues barely affected the assembly and maturation of 40S subunits and the initiation of translation, but completely prevented the formation of polysomes. This implied the crucial importance of the uS19 tail in the elongation process. Analysis of tRNAs associated with 40S subunits and 80S ribosomes containing wild type uS19FLAG or its truncated form showed that the deletion of the C-terminal pentadecapeptide fragment of uS19 did not interfere with the binding of aminoacyl-tRNA (aa-tRNA) at the ribosomal A site. The results led to the conclusion that the transpeptidation, which occurs on the large ribosomal subunit after decoding the A site codon by the incoming aa-tRNA, is the most likely elongation stage, where this uS19 fragment can play a critical role. Our findings suggest that the uS19 tail is a keystone player in the accommodation of aa-tRNA at the A site, which is a pre-requisite for the peptide transfer.


Assuntos
Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Células HEK293 , Humanos , Polirribossomos/metabolismo , RNA de Transferência/metabolismo , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Deleção de Sequência
10.
Genes Dev ; 34(1-2): 118-131, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31831626

RESUMO

Small RNAs derived from mature tRNAs, referred to as tRNA fragments or "tRFs," are an emerging class of regulatory RNAs with poorly understood functions. We recently identified a role for one specific tRF-5' tRF-Gly-GCC, or tRF-GG-as a repressor of genes associated with the endogenous retroelement MERVL, but the mechanistic basis for this regulation was unknown. Here, we show that tRF-GG plays a role in production of a wide variety of noncoding RNAs-snoRNAs, scaRNAs, and snRNAs-that are dependent on Cajal bodies for stability and activity. Among these noncoding RNAs, regulation of the U7 snRNA by tRF-GG modulates heterochromatin-mediated transcriptional repression of MERVL elements by supporting an adequate supply of histone proteins. Importantly, the effects of inhibiting tRF-GG on histone mRNA levels, on activity of a histone 3' UTR reporter, and ultimately on MERVL regulation could all be suppressed by manipulating U7 RNA levels. We additionally show that the related RNA-binding proteins hnRNPF and hnRNPH bind directly to tRF-GG, and are required for Cajal body biogenesis, positioning these proteins as strong candidates for effectors of tRF-GG function in vivo. Together, our data reveal a conserved mechanism for 5' tRNA fragment control of noncoding RNA biogenesis and, consequently, global chromatin organization.


Assuntos
Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , RNA de Transferência/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Animais , Linhagem Celular , Corpos Enovelados/metabolismo , Células-Tronco Embrionárias Humanas , Humanos , Camundongos , Ligação Proteica , RNA Nuclear Pequeno/genética , Retroelementos/genética
11.
Proc Natl Acad Sci U S A ; 117(2): 982-992, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879344

RESUMO

The exoribonuclease Rrp6p is critical for RNA decay in the nucleus. While Rrp6p acts on a large range of diverse substrates, it does not indiscriminately degrade all RNAs. How Rrp6p accomplishes this task is not understood. Here, we measure Rrp6p-RNA binding and degradation kinetics in vitro at single-nucleotide resolution and find an intrinsic substrate selectivity that enables Rrp6p to discriminate against specific RNAs. RNA length and the four 3'-terminal nucleotides contribute most to substrate selectivity and collectively enable Rrp6p to discriminate between different RNAs by several orders of magnitude. The most pronounced discrimination is seen against RNAs ending with CCA-3'. These RNAs correspond to 3' termini of uncharged tRNAs, which are not targeted by Rrp6p in cells. The data show that in contrast to many other proteins that use substrate selectivity to preferentially interact with specific RNAs, Rrp6p utilizes its selectivity to discriminate against specific RNAs. This ability allows Rrp6p to target diverse substrates while avoiding a subset of RNAs.


Assuntos
Exorribonucleases/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Escherichia coli , Exorribonucleases/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Cinética , RNA/química , RNA de Transferência/metabolismo , Especificidade por Substrato
12.
Nat Struct Mol Biol ; 27(1): 25-32, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31873307

RESUMO

The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for ribosome recycling by RRF and EF-G remains unclear. Here, we report the crystal structure of a posttermination Thermus thermophilus 70S ribosome complexed with EF-G, RRF and two transfer RNAs at a resolution of 3.5 Å. The deacylated tRNA in the peptidyl (P) site moves into a previously unsuspected state of binding (peptidyl/recycling, p/R) that is analogous to that seen during initiation. The terminal end of the p/R-tRNA forms nonfavorable contacts with the 50S subunit while RRF wedges next to central inter-subunit bridges, illuminating the active roles of tRNA and RRF in dissociation of ribosomal subunits. The structure uncovers a missing snapshot of tRNA as it transits between the P and exit (E) sites, providing insights into the mechanisms of ribosome recycling and tRNA translocation.


Assuntos
Proteínas de Bactérias/metabolismo , RNA de Transferência/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Thermus thermophilus/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Modelos Moleculares , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Conformação Proteica , RNA de Transferência/química , Proteínas Ribossômicas/química , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Ribossomos/química , Thermus thermophilus/química
13.
Nat Chem Biol ; 16(3): 310-317, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31844301

RESUMO

Chloramphenicol (CHL) and linezolid (LZD) are antibiotics that inhibit translation. Both were thought to block peptide-bond formation between all combinations of amino acids. Yet recently, a strong nascent peptide context-dependency of CHL- and LZD-induced translation arrest was discovered. Here we probed the mechanism of action of CHL and LZD by using single-molecule Förster resonance energy transfer spectroscopy to monitor translation arrest induced by antibiotics. The presence of CHL or LZD does not substantially alter dynamics of protein synthesis until the arrest-motif of the nascent peptide is generated. Inhibition of peptide-bond formation compels the fully accommodated A-site transfer RNA to undergo repeated rounds of dissociation and nonproductive rebinding. The glycyl amino-acid moiety on the A-site Gly-tRNA manages to overcome the arrest by CHL. Our results illuminate the mechanism of CHL and LZD action through their interactions with the ribosome, the nascent peptide and the incoming amino acid, perturbing elongation dynamics.


Assuntos
Cloranfenicol/farmacologia , Linezolida/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Aminoácidos/metabolismo , Antibacterianos/farmacologia , Sítios de Ligação , Cloranfenicol/metabolismo , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Linezolida/metabolismo , Peptídeos/metabolismo , Ligação Proteica , RNA de Transferência/metabolismo , Ribossomos/metabolismo
14.
Nat Commun ; 10(1): 5774, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852903

RESUMO

Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.


Assuntos
Códon/genética , Elongação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Aminoácidos/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Biblioteca Gênica , Genes Reporter/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nucleotídeos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas RGS/genética , Proteínas RGS/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula
15.
Nat Struct Mol Biol ; 26(12): 1132-1140, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31768042

RESUMO

Faulty or damaged messenger RNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein degradation and ribosome recycling. The most common mRNA defect in eukaryotes is probably inappropriate polyadenylation at near-cognate sites within the coding region. How ribosomes stall selectively when they encounter poly(A) is unclear. Here, we use biochemical and structural approaches in mammalian systems to show that poly-lysine, encoded by poly(A), favors a peptidyl-transfer RNA conformation suboptimal for peptide bond formation. This conformation partially slows elongation, permitting poly(A) mRNA in the ribosome's decoding center to adopt a ribosomal RNA-stabilized single-stranded helix. The reconfigured decoding center clashes with incoming aminoacyl-tRNA, thereby precluding elongation. Thus, coincidence detection of poly-lysine in the exit tunnel and poly(A) in the decoding center allows ribosomes to detect aberrant mRNAs selectively, stall elongation and trigger downstream quality control pathways essential for cellular homeostasis.


Assuntos
Peptídeos/metabolismo , Poli A/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Peptídeos/química , Poli A/química , Poliadenilação , Polilisina/química , Polilisina/metabolismo , Estabilidade de RNA , RNA Mensageiro/química , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/química
16.
Nat Struct Mol Biol ; 26(12): 1106-1113, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740853

RESUMO

T-box riboregulators are a class of cis-regulatory RNAs that govern the bacterial response to amino acid starvation by binding, decoding and reading the aminoacylation status of specific transfer RNAs. Here we provide a high-resolution crystal structure of a full-length T-box from Mycobacterium tuberculosis that explains tRNA decoding and aminoacylation sensing by this riboregulator. Overall, the T-box consists of decoding and aminoacylation sensing modules bridged by a rigid pseudoknot structure formed by the mid-region domains. Stem-I and the Stem-II S-turn assemble a claw-like decoding module, while the antiterminator, Stem-III, and the adjacent linker form a tightly interwoven aminoacylation sensing module. The uncharged tRNA is selectively recognized by an unexpected set of favorable contacts from the linker region in the aminoacylation sensing module. A complex structure with a charged tRNA mimic shows that the extra moiety dislodges the linker, which is indicative of the possible chain of events that lead to alternative base-pairing and altered expression output.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Proteínas com Domínio T/metabolismo , Aminoacilação , Proteínas de Bactérias/química , Pareamento de Bases , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/química , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA de Transferência/química , Riboswitch , Proteínas com Domínio T/química , Tuberculose/microbiologia
17.
Nat Struct Mol Biol ; 26(12): 1094-1105, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740854

RESUMO

Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report cocrystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box-tRNA complexes, detailing their multivalent, exquisitely selective interactions. The T-box forms a U-shaped molecular vise that clamps the tRNA, captures its 3' end using an elaborate 'discriminator' structure, and interrogates its aminoacylation state using a steric filter fashioned from a wobble base pair. In the absence of aminoacylation, T-boxes clutch tRNAs and form a continuously stacked central spine, permitting transcriptional readthrough or translation initiation. A modeled aminoacyl disrupts tRNA-T-box stacking, severing the central spine and blocking gene expression. Our data establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches and exemplify how higher-order RNA-RNA interactions achieve multivalency and specificity.


Assuntos
Aminoácidos/metabolismo , Bacillus subtilis/metabolismo , Geobacillus/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Riboswitch , Aminoacilação , Bacillus subtilis/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Geobacillus/química , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/ultraestrutura , RNA de Transferência/química , RNA de Transferência/ultraestrutura
18.
Nat Commun ; 10(1): 4598, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601802

RESUMO

mRNA contexts containing a 'slippery' sequence and a downstream secondary structure element stall the progression of the ribosome along the mRNA and induce its movement into the -1 reading frame. In this study we build a thermodynamic model based on Bayesian statistics to explain how -1 programmed ribosome frameshifting can work. As training sets for the model, we measured frameshifting efficiencies on 64 dnaX mRNA sequence variants in vitro and also used 21 published in vivo efficiencies. With the obtained free-energy difference between mRNA-tRNA base pairs in the 0 and -1 frames, the frameshifting efficiency of a given sequence can be reproduced and predicted from the tRNA-mRNA base pairing in the two frames. Our results further explain how modifications in the tRNA anticodon modulate frameshifting and show how the ribosome tunes the strength of the base-pair interactions.


Assuntos
Proteínas de Bactérias/genética , DNA Polimerase III/genética , Mudança da Fase de Leitura do Gene Ribossômico/fisiologia , Modelos Teóricos , Pareamento de Bases , Teorema de Bayes , Códon , Mutação da Fase de Leitura , Lisina/genética , Fenilalanina/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Termodinâmica
19.
Mol Cells ; 42(10): 687-692, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31656062

RESUMO

Transfer RNA-derived small RNAs (tsRNAs) play a role in various cellular processes. Accumulating evidence has revealed that tsRNAs are deeply implicated in human diseases, such as various cancers and neurological disorders, suggesting that tsRNAs should be investigated to develop novel therapeutic intervention. tsRNAs provide more complexity to the physiological role of transfer RNAs by repressing or activating protein synthesis with distinct mechanisms. Here, we highlight the detailed mechanism of tsRNA-mediated dual regulation in protein synthesis and discuss the necessity of novel sequencing technology to learn more about tsRNAs.


Assuntos
Biossíntese de Proteínas , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Animais , Humanos , Ribossomos/metabolismo , Estresse Fisiológico
20.
Biochem Soc Trans ; 47(5): 1481-1488, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31647517

RESUMO

Cells must be able to sense and adapt to their surroundings to thrive in a dynamic environment. Key to adapting to a low nutrient environment is the Integrated Stress Response (ISR), a short-lived pathway that allows cells to either regain cellular homeostasis or facilitate apoptosis during periods of stress. Central to the ISR is the protein kinase General Control Non-depressible 2 (GCN2), which is responsible for sensing starvation. Upon amino acid deficiency, GCN2 is activated and initiates the ISR by phosphorylating the translation initiation factor eIF2α, stalling protein translation, and activating the transcription factor ATF4, which in turn up-regulates autophagy and biosynthesis pathways. A key outstanding question is how GCN2 is activated from an autoinhibited state. Until recently, a model of activation focussed on the increase of deacylated tRNA associated with amino acid starvation, with deacylated tRNA binding directly to GCN2 and releasing autoinhibition. However, in vivo experiments have pointed towards an alternative, deacylated-tRNA-independent mechanism of activation. Here, we review the various factors that may facilitate GCN2 activation, including recent research showing that the P-stalk complex, a ribosome-associated heteropentameric protein complex, is a potent activator of GCN2.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Ativação Enzimática , Humanos , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , RNA de Transferência/metabolismo
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