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1.
Cell ; 186(13): 2929-2949.e20, 2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37269831

RESUMO

Lifespan varies within and across species, but the general principles of its control remain unclear. Here, we conducted multi-tissue RNA-seq analyses across 41 mammalian species, identifying longevity signatures and examining their relationship with transcriptomic biomarkers of aging and established lifespan-extending interventions. An integrative analysis uncovered shared longevity mechanisms within and across species, including downregulated Igf1 and upregulated mitochondrial translation genes, and unique features, such as distinct regulation of the innate immune response and cellular respiration. Signatures of long-lived species were positively correlated with age-related changes and enriched for evolutionarily ancient essential genes, involved in proteolysis and PI3K-Akt signaling. Conversely, lifespan-extending interventions counteracted aging patterns and affected younger, mutable genes enriched for energy metabolism. The identified biomarkers revealed longevity interventions, including KU0063794, which extended mouse lifespan and healthspan. Overall, this study uncovers universal and distinct strategies of lifespan regulation within and across species and provides tools for discovering longevity interventions.


Assuntos
Longevidade , Fosfatidilinositol 3-Quinases , Animais , Camundongos , Longevidade/genética , Fosfatidilinositol 3-Quinases/genética , Envelhecimento/genética , Mamíferos/genética , Perfilação da Expressão Gênica
2.
Semin Cell Dev Biol ; 154(Pt B): 138-154, 2024 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-37357122

RESUMO

Cellular stress is an intrinsic part of cell physiology that underlines cell survival or death. The ability of mammalian cells to regulate global protein synthesis (aka translational control) represents a critical, yet underappreciated, layer of regulation during the stress response. Various cellular stress response pathways monitor conditions of cell growth and subsequently reshape the cellular translatome to optimize translational outputs. On the molecular level, such translational reprogramming involves an intricate network of interactions between translation machinery, RNA-binding proteins, mRNAs, and non-protein coding RNAs. In this review, we will discuss molecular mechanisms, signaling pathways, and targets of translational control that contribute to cellular adaptation to stress and to cell survival or death.


Assuntos
Biossíntese de Proteínas , Transdução de Sinais , Animais , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Morte Celular , Mamíferos/genética
3.
Mol Cell ; 71(5): 761-774.e5, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30146315

RESUMO

The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo. Ribosome profiling of tma deletion strains revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3' UTR, as evidenced by peaks in the footprint data and 3' UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream open reading frames (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an 80S reinitiation process in 3' UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in recycling 40S ribosomal subunits at stop codons and translation reinitiation.


Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Subunidades Ribossômicas Menores de Eucariotos/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas/genética , Códon de Terminação/genética , Escherichia coli/genética , Fases de Leitura Aberta/genética , Iniciação Traducional da Cadeia Peptídica/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética
4.
Biochemistry (Mosc) ; 89(2): 313-321, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38622098

RESUMO

AgeMeta is a database that provides systemic and quantitative description of mammalian aging at the level of gene expression. It encompasses transcriptomic changes with age across various tissues of humans, mice, and rats, based on a comprehensive meta-analysis of 122 publicly available gene expression datasets from 26 studies. AgeMeta provides an intuitive visual interface for quantification of aging-associated transcriptomics at the level of individual genes and functional groups of genes, allowing easy comparison among various species and tissues. Additionally, all the data in the database can be downloaded and analyzed independently. Overall, this work contributes to the understanding of the complex network of biological processes underlying mammalian aging and supports future advancements in this field. AgeMeta is freely available at: https://age-meta.com/.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Ratos , Camundongos , Humanos , Animais , Envelhecimento/genética , Bases de Dados Factuais , Mamíferos/genética
5.
Mol Biol Evol ; 39(1)2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34905062

RESUMO

In most eukaryotic genomes, tandemly repeated copies of 5S rRNA genes are clustered outside the nucleolus organizer region (NOR), which normally encodes three other major rRNAs: 18S, 5.8S, and 28S. Our analysis of turtle rDNA sequences has revealed a 5S rDNA insertion into the NOR intergenic spacer in antisense orientation. The insertion (hereafter called NOR-5S rRNA gene) has a length of 119 bp and coexists with the canonical 5S rDNA clusters outside the NOR. Despite the ∼20% nucleotide difference between the two 5S gene sequences, their internal control regions for RNA polymerase III are similar. Using the turtle Trachemys scripta as a model species, we showed the NOR-5S rDNA specific expression in oocytes. This expression is concurrent with the NOR rDNA amplification during oocyte growth. We show that in vitellogenic oocytes, the NOR-5S rRNA prevails over the canonical 5S rRNA in the ribosomes, suggesting a role of modified ribosomes in oocyte-specific translation. The orders Testudines and Crocodilia seem to be the only taxa of vertebrates with such a peculiar rDNA organization. We speculate that the amplification of the 5S rRNA genes as a part of the NOR DNA during oogenesis provides a dosage balance between transcription of all the four ribosomal RNAs while producing a maternal pool of extra ribosomes. We further hypothesize that the NOR-5S rDNA insertion appeared in the Archelosauria clade during the Permian period and was lost later in the ancestors of Aves.


Assuntos
Jacarés e Crocodilos , Tartarugas , Jacarés e Crocodilos/genética , Animais , DNA Ribossômico/genética , Genes de RNAr , Oócitos , RNA Ribossômico 5S/genética , Tartarugas/genética
6.
Biochemistry (Mosc) ; 88(11): 1786-1799, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38105199

RESUMO

In response to stress stimuli, eukaryotic cells typically suppress protein synthesis. This leads to the release of mRNAs from polysomes, their condensation with RNA-binding proteins, and the formation of non-membrane-bound cytoplasmic compartments called stress granules (SGs). SGs contain 40S but generally lack 60S ribosomal subunits. It is known that cycloheximide, emetine, and anisomycin, the ribosome inhibitors that block the progression of 80S ribosomes along mRNA and stabilize polysomes, prevent SG assembly. Conversely, puromycin, which induces premature termination, releases mRNA from polysomes and stimulates the formation of SGs. The same effect is caused by some translation initiation inhibitors, which lead to polysome disassembly and the accumulation of mRNAs in the form of stalled 48S preinitiation complexes. Based on these and other data, it is believed that the trigger for SG formation is the presence of mRNA with extended ribosome-free segments, which tend to form condensates in the cell. In this study, we evaluated the ability of various small-molecule translation inhibitors to block or stimulate the assembly of SGs under conditions of severe oxidative stress induced by sodium arsenite. Contrary to expectations, we found that ribosome-targeting elongation inhibitors of a specific type, which arrest solitary 80S ribosomes at the beginning of the mRNA coding regions but do not interfere with all subsequent ribosomes in completing translation and leaving the transcripts (such as harringtonine, lactimidomycin, or T-2 toxin), completely prevent the formation of arsenite-induced SGs. These observations suggest that the presence of even a single 80S ribosome on mRNA is sufficient to prevent its recruitment into SGs, and the presence of extended ribosome-free regions of mRNA is not sufficient for SG formation. We propose that mRNA entry into SGs may be mediated by specific contacts between RNA-binding proteins and those regions on 40S subunits that remain inaccessible when ribosomes are associated.


Assuntos
Biossíntese de Proteínas , Grânulos de Estresse , RNA Mensageiro/metabolismo , Grânulos Citoplasmáticos , Ribossomos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas de Ligação a RNA/metabolismo
7.
Nucleic Acids Res ; 49(19): 11134-11144, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34606617

RESUMO

The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the 'head-to-head' orientation of the deleted and neighboring genes, knockout often led to a shift of the transcription start site of the latter, introducing new uAUG codon(s) into the expanded 5' untranslated region (5' UTR). In the 'tail-to-tail' arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3' UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as ∼1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.


Assuntos
Loci Gênicos/efeitos dos fármacos , Gentamicinas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Deleção de Sequência , Transcrição Gênica/efeitos dos fármacos , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Bases , Códon , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes/métodos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fases de Leitura Aberta , Ribossomos/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sítio de Iniciação de Transcrição
8.
Proc Natl Acad Sci U S A ; 117(27): 15581-15590, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576685

RESUMO

Protein synthesis represents a major metabolic activity of the cell. However, how it is affected by aging and how this in turn impacts cell function remains largely unexplored. To address this question, herein we characterized age-related changes in both the transcriptome and translatome of mouse tissues over the entire life span. We showed that the transcriptome changes govern those in the translatome and are associated with altered expression of genes involved in inflammation, extracellular matrix, and lipid metabolism. We also identified genes that may serve as candidate biomarkers of aging. At the translational level, we uncovered sustained down-regulation of a set of 5'-terminal oligopyrimidine (5'-TOP) transcripts encoding protein synthesis and ribosome biogenesis machinery and regulated by the mTOR pathway. For many of them, ribosome occupancy dropped twofold or even more. Moreover, with age, ribosome coverage gradually decreased in the vicinity of start codons and increased near stop codons, revealing complex age-related changes in the translation process. Taken together, our results reveal systematic and multidimensional deregulation of protein synthesis, showing how this major cellular process declines with age.


Assuntos
Envelhecimento/fisiologia , Regulação da Expressão Gênica/fisiologia , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Animais , Códon de Iniciação/metabolismo , Biologia Computacional , Masculino , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq , Ribossomos/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma/fisiologia
9.
Int J Mol Sci ; 24(9)2023 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-37176068

RESUMO

While protein synthesis is vital for the majority of cell types of the human body, diversely differentiated cells require specific translation regulation. This suggests the specialization of translation machinery across tissues and organs. Using transcriptomic data from GTEx, FANTOM, and Gene Atlas, we systematically explored the abundance of transcripts encoding translation factors and aminoacyl-tRNA synthetases (ARSases) in human tissues. We revised a few known and identified several novel translation-related genes exhibiting strict tissue-specific expression. The proteins they encode include eEF1A1, eEF1A2, PABPC1L, PABPC3, eIF1B, eIF4E1B, eIF4ENIF1, and eIF5AL1. Furthermore, our analysis revealed a pervasive tissue-specific relative abundance of translation machinery components (e.g., PABP and eRF3 paralogs, eIF2B and eIF3 subunits, eIF5MPs, and some ARSases), suggesting presumptive variance in the composition of translation initiation, elongation, and termination complexes. These conclusions were largely confirmed by the analysis of proteomic data. Finally, we paid attention to sexual dimorphism in the repertoire of translation factors encoded in sex chromosomes (eIF1A, eIF2γ, and DDX3), and identified the testis and brain as organs with the most diverged expression of translation-associated genes.


Assuntos
Aminoacil-tRNA Sintetases , Proteômica , Humanos , Fatores de Iniciação de Peptídeos , Fator 1 de Elongação de Peptídeos
10.
Nat Chem Biol ; 16(10): 1071-1077, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32601485

RESUMO

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.


Assuntos
Amycolatopsis/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Amycolatopsis/genética , Amycolatopsis/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Farmacorresistência Bacteriana , Escherichia coli , Células HEK293 , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutação , Naftacenos/química , Naftacenos/farmacologia , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , Ribossomos/metabolismo
11.
Proc Natl Acad Sci U S A ; 116(2): 528-533, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30584092

RESUMO

The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. We determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR's MCT-1-binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.


Assuntos
Proteínas de Ciclo Celular/química , Fatores de Iniciação em Eucariotos/química , Proteínas Oncogênicas/química , Multimerização Proteica , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Estrutura Quaternária de Proteína
12.
Biochemistry (Mosc) ; 86(9): 1060-1094, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34565312

RESUMO

Viruses exploit the translation machinery of an infected cell to synthesize their proteins. Therefore, viral mRNAs have to compete for ribosomes and translation factors with cellular mRNAs. To succeed, eukaryotic viruses adopt multiple strategies. One is to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment. These include internal ribosome entry sites (IRESs), which make translation independent of the free 5' end, or cap-independent translational enhancers (CITEs), which promote initiation at the uncapped 5' end, even if located in 3' untranslated regions (3' UTRs). Even if a virus uses the canonical cap-dependent ribosome recruitment, it can still perturb conventional ribosomal scanning and start codon selection. The pressure for genome compression often gives rise to internal and overlapping open reading frames. Their translation is initiated through specific mechanisms, such as leaky scanning, 43S sliding, shunting, or coupled termination-reinitiation. Deviations from the canonical initiation reduce the dependence of viral mRNAs on translation initiation factors, thereby providing resistance to antiviral mechanisms and cellular stress responses. Moreover, viruses can gain advantage in a competition for the translational machinery by inactivating individual translational factors and/or replacing them with viral counterparts. Certain viruses even create specialized intracellular "translation factories", which spatially isolate the sites of their protein synthesis from cellular antiviral systems, and increase availability of translational components. However, these virus-specific mechanisms may become the Achilles' heel of a viral life cycle. Thus, better understanding of the unconventional mechanisms of viral mRNA translation initiation provides valuable insight for developing new approaches to antiviral therapy.


Assuntos
Células Eucarióticas/virologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Animais , Células Eucarióticas/fisiologia , Humanos , Sítios Internos de Entrada Ribossomal/fisiologia , RNA Circular/genética , Proteínas Virais/fisiologia
13.
Int J Mol Sci ; 21(5)2020 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-32121426

RESUMO

The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5' and 3' ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or m7GpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5' untranslated region (UTR) and 3' UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.


Assuntos
Iniciação Traducional da Cadeia Peptídica/genética , Poli A/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/química , Regiões 3' não Traduzidas/genética , Animais , Sistema Livre de Células , Ciclização , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação Eucariótico 4G/química , Fator de Iniciação Eucariótico 4G/genética , Camundongos , Poli A/química , Capuzes de RNA/química , Capuzes de RNA/genética
14.
BMC Bioinformatics ; 20(1): 113, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30841857

RESUMO

BACKGROUND: High-throughput sequencing often provides a foundation for experimental analyses in the life sciences. For many such methods, an intermediate layer of bioinformatics data analysis is the genomic signal track constructed by short read mapping to a particular genome assembly. There are many software tools to visualize genomic tracks in a web browser or with a stand-alone graphical user interface. However, there are only few command-line applications suitable for automated usage or production of publication-ready visualizations. RESULTS: Here we present svist4get, a command-line tool for customizable generation of publication-quality figures based on data from genomic signal tracks. Similarly to generic genome browser software, svist4get visualizes signal tracks at a given genomic location and is able to aggregate data from several tracks on a single plot along with the transcriptome annotation. The resulting plots can be saved as the vector or high-resolution bitmap images. We demonstrate practical use cases of svist4get for Ribo-Seq and RNA-Seq data. CONCLUSIONS: svist4get is implemented in Python 3 and runs on Linux. The command-line interface of svist4get allows for easy integration into bioinformatics pipelines in a console environment. Extra customization is possible through configuration files and Python API. For convenience, svist4get is provided as pypi package. The source code is available at https://bitbucket.org/artegorov/svist4get/.


Assuntos
Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Software , Genoma , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
15.
Nucleic Acids Res ; 45(2): 513-526, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-27923997

RESUMO

The development of Ribosome Profiling (RiboSeq) has revolutionized functional genomics. RiboSeq is based on capturing and sequencing of the mRNA fragments enclosed within the translating ribosome and it thereby provides a 'snapshot' of ribosome positions at the transcriptome wide level. Although the method is predominantly used for analysis of differential gene expression and discovery of novel translated ORFs, the RiboSeq data can also be a rich source of information about molecular mechanisms of polypeptide synthesis and translational control. This review will focus on how recent findings made with RiboSeq have revealed important details of the molecular mechanisms of translation in eukaryotes. These include mRNA translation sensitivity to drugs affecting translation initiation and elongation, the roles of upstream ORFs in response to stress, the dynamics of elongation and termination as well as details of intrinsic ribosome behavior on the mRNA after translation termination. As the RiboSeq method is still at a relatively early stage we will also discuss the implications of RiboSeq artifacts on data interpretation.


Assuntos
Biossíntese de Proteínas , Ribossomos/metabolismo , Animais , Códon de Iniciação , Regulação da Expressão Gênica , Humanos , Complexos Multiproteicos , Fases de Leitura Aberta , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Subunidades Ribossômicas/metabolismo
16.
Cell Mol Life Sci ; 74(8): 1431-1455, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27853833

RESUMO

The idea of internal initiation is frequently exploited to explain the peculiar translation properties or unusual features of some eukaryotic mRNAs. In this review, we summarize the methods and arguments most commonly used to address cases of translation governed by internal ribosome entry sites (IRESs). Frequent mistakes are revealed. We explain why "cap-independent" does not readily mean "IRES-dependent" and why the presence of a long and highly structured 5' untranslated region (5'UTR) or translation under stress conditions cannot be regarded as an argument for appealing to internal initiation. We carefully describe the known pitfalls and limitations of the bicistronic assay and artefacts of some commercially available in vitro translation systems. We explain why plasmid DNA transfection should not be used in IRES studies and which control experiments are unavoidable if someone decides to use it anyway. Finally, we propose a workflow for the validation of IRES activity, including fast and simple experiments based on a single genetic construct with a sequence of interest.


Assuntos
Sítios Internos de Entrada Ribossomal , Regiões 5' não Traduzidas , Animais , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transfecção
17.
Nucleic Acids Res ; 44(4): 1882-93, 2016 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-26717981

RESUMO

During eukaryotic translation initiation, 43S ribosomal complex scans mRNA leader unless an AUG codon in an appropriate context is found. Establishing the stable codon-anticodon base-pairing traps the ribosome on the initiator codon and triggers structural rearrangements, which lead to Pi release from the eIF2-bound GTP. It is generally accepted that AUG recognition by the scanning 43S complex sets the final point in the process of start codon selection, while latter stages do not contribute to this process. Here we use translation reconstitution approach and kinetic toe-printing assay to show that after the 48S complex is formed on an AUG codon, in case GTP hydrolysis is impaired, the ribosomal subunit is capable to resume scanning and slides downstream to the next AUG. In contrast to leaky scanning, this sliding is not limited to AUGs in poor nucleotide contexts and occurs after a relatively long pause at the recognized AUG. Thus, recognition of an AUG per se does not inevitably lead to this codon being selected for initiation of protein synthesis. Instead, it is eIF5-induced GTP hydrolysis and Pi release that irreversibly trap the 48S complex, and this complex is further stabilized by eIF5B and 60S joining.


Assuntos
Fator de Iniciação 2 em Eucariotos/genética , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Anticódon/genética , Códon/genética , Escherichia coli , Fator de Iniciação 2 em Eucariotos/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólise , Cinética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A
18.
Biochim Biophys Acta ; 1859(10): 1259-68, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27424222

RESUMO

Signaling lymphocytic activation molecule family member 1 (SLAMF1)/CD150 is a co-stimulatory receptor expressed on a variety of hematopoietic cells, in particular on mature lymphocytes activated by specific antigen, costimulation and cytokines. Changes in CD150 expression level have been reported in association with autoimmunity and with B-cell chronic lymphocytic leukemia. We characterized the core promoter for SLAMF1 gene in human B-cell lines and explored binding sites for a number of transcription factors involved in B cell differentiation and activation. Mutations of SP1, STAT6, IRF4, NF-kB, ELF1, TCF3, and SPI1/PU.1 sites resulted in significantly decreased promoter activity of varying magnitude, depending on the cell line tested. The most profound effect on the promoter strength was observed upon mutation of the binding site for Early B-cell factor 1 (EBF1). This mutation produced a 10-20 fold drop in promoter activity and pinpointed EBF1 as the master regulator of human SLAMF1 gene in B cells. We also identified three potent transcriptional enhancers in human SLAMF1 locus, each containing functional EBF1 binding sites. Thus, EBF1 interacts with specific binding sites located both in the promoter and in the enhancer regions of the SLAMF1 gene and is critical for its expression in human B cells.


Assuntos
Regulação da Expressão Gênica , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Transativadores/genética , Transcrição Gênica , Linfócitos B/citologia , Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos , Genes Reporter , Células HEK293 , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Luciferases/genética , Mutação , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Crit Rev Biochem Mol Biol ; 49(2): 164-77, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24520918

RESUMO

The conventional paradigm of translation initiation in eukaryotes states that the cap-binding protein complex eIF4F (consisting of eIF4E, eIF4G and eIF4A) plays a central role in the recruitment of capped mRNAs to ribosomes. However, a growing body of evidence indicates that this paradigm should be revised. This review summarizes the data which have been mostly accumulated in a post-genomic era owing to revolutionary techniques of transcriptome-wide analysis. Unexpectedly, these techniques have uncovered remarkable diversity in the recruitment of cellular mRNAs to eukaryotic ribosomes. These data enable a preliminary classification of mRNAs into several groups based on their requirement for particular components of eIF4F. They challenge the widely accepted concept which relates eIF4E-dependence to the extent of secondary structure in the 5' untranslated regions of mRNAs. Moreover, some mRNA species presumably recruit ribosomes to their 5' ends without the involvement of either the 5' m(7)G-cap or eIF4F but instead utilize eIF4G or eIF4G-like auxiliary factors. The long-standing concept of internal ribosome entry site (IRES)-elements in cellular mRNAs is also discussed.


Assuntos
RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Transcriptoma , Animais , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/genética , Ribossomos/química , Ribossomos/genética
20.
Nucleic Acids Res ; 42(20): 12779-88, 2014 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-25326319

RESUMO

TRPM7 is an essential and ubiquitous channel-kinase regulating cellular influx of Mg2+. Although TRPM7 mRNA is highly abundant, very small amount of the protein is detected in cells, suggesting post-transcriptional regulation of trpm7 gene expression. We found that TRPM7 mRNA 5'-leader contains two evolutionarily conserved upstream open reading frames that act together to drastically inhibit translation of the TRPM7 reading frame at high magnesium levels and ensure its optimal translation at low magnesium levels, when the activity of the channel-kinase is most required. The study provides the first example of magnesium channel synthesis being controlled by Mg2+ in higher eukaryotes.


Assuntos
Regiões 5' não Traduzidas , Regulação da Expressão Gênica , Magnésio/farmacologia , Fases de Leitura Aberta , Biossíntese de Proteínas , Canais de Cátion TRPM/genética , Sequência de Bases , Sequência Conservada , Células HEK293 , Humanos , Biossíntese de Proteínas/efeitos dos fármacos
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