RESUMO
The synthesis of most proteins begins at AUG codons, yet a small number of non-AUG initiated proteoforms are also known. Here we analyse a large number of publicly available Ribo-seq datasets to identify novel, previously uncharacterised non-AUG proteoforms using Trips-Viz implementation of a novel algorithm for detecting translated ORFs. In parallel we analyse genomic alignment of 120 mammals to identify evidence of protein coding evolution in sequences encoding potential extensions. Unexpectedly we find that the number of non-AUG proteoforms identified with ribosome profiling data greatly exceeds those with strong phylogenetic support suggesting their recent evolution. Our study argues that the protein coding potential of human genome greatly exceeds that detectable through comparative genomics and exposes the existence of multiple proteins encoded by the same genomic loci.
Assuntos
Genômica , Ribossomos , Animais , Humanos , Ribossomos/metabolismo , Filogenia , Códon/genética , Códon/metabolismo , Proteínas/metabolismo , Biossíntese de Proteínas , Fases de Leitura Aberta/genética , Mamíferos/genética , Mamíferos/metabolismoRESUMO
The phenomenon of internal initiation of translation was discovered in 1988 on poliovirus mRNA. The prototypic cis-acting element in the 5' untranslated region (5'UTR) of poliovirus mRNA, which is able to direct initiation at an internal start codon without the involvement of a cap structure, has been called an IRES (Internal Ribosome Entry Site or Segment). Despite its early discovery, poliovirus and other related IRES elements of type I are poorly characterized, and it is not yet clear which host proteins (a.k.a. IRES trans-acting factors, ITAFs) are required for their full activity in vivo. Here we discuss recent and old results devoted to type I IRESes and provide evidence that Poly(rC) binding protein 2 (PCBP2), Glycyl-tRNA synthetase (GARS), and Cold Shock Domain Containing E1 (CSDE1, also known as UNR) are major regulators of type I IRES activity.
Assuntos
Poliovirus , Poliovirus/genética , Poliovirus/metabolismo , Sítios Internos de Entrada Ribossomal/genética , Transativadores/metabolismo , Sequências Reguladoras de Ácido Nucleico , Códon de Iniciação/metabolismo , RNA Mensageiro/metabolismo , Biossíntese de Proteínas , Regiões 5' não Traduzidas , RNA Viral/metabolismoRESUMO
Activated ghrelin receptor GHS-R1α triggers cell signalling pathways that modulate energy homeostasis and biosynthetic processes. However, the effects of ghrelin on mRNA translation are unknown. Using various reporter assays, here we demonstrate a rapid elevation of protein synthesis in cells within 15-30 min upon stimulation of GHS-R1α by ghrelin. We further show that ghrelin-induced activation of translation is mediated, at least in part, through the de-phosphorylation (de-suppression) of elongation factor 2 (eEF2). The levels of eEF2 phosphorylation at Thr56 decrease due to the reduced activity of eEF2 kinase, which is inhibited via Ser366 phosphorylation by rpS6 kinases. Being stress-susceptible, the ghrelin-mediated decrease in eEF2 phosphorylation can be abolished by glucose deprivation and mitochondrial uncoupling. We believe that the observed burst of translation benefits rapid restocking of neuropeptides, which are released upon GHS-R1α activation, and represents the most time- and energy-efficient way of prompt recharging the orexigenic neuronal circuitry.
Assuntos
Grelina , Biossíntese de Proteínas , Grelina/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Fosforilação , Transdução de Sinais/fisiologiaRESUMO
Recent proteogenomic studies revealed extensive translation outside of annotated protein coding regions, such as non-coding RNAs and untranslated regions of mRNAs. This non-canonical translation is largely due to start codon plurality within the same RNA. This plurality is often due to the failure of some scanning ribosomes to recognize potential start codons leading to initiation downstream-a process termed leaky scanning. Codons other than AUG (non-AUG) are particularly leaky due to their inefficiency. Here we discuss our current understanding of non-AUG initiation. We argue for a near-ubiquitous role of non-AUG initiation in shaping the dynamic composition of mammalian proteomes.
Assuntos
Mamíferos , Ribossomos , Animais , Códon , Códon de Iniciação/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Mutations in genes encoding cytochrome c oxidase (mitochondrial complex IV) subunits and assembly factors [e.g., synthesis of cytochrome c oxidase 2 (SCO2)] are linked to severe metabolic syndromes. Notwithstanding that SCO2 is under transcriptional control of tumor suppressor p53, the role of mitochondrial complex IV dysfunction in cancer metabolism remains obscure. Herein, we demonstrate that the loss of SCO2 in HCT116 colorectal cancer cells leads to significant metabolic and signaling perturbations. Specifically, abrogation of SCO2 increased NAD+ regenerating reactions and decreased glucose oxidation through citric acid cycle while enhancing pyruvate carboxylation. This was accompanied by a reduction in amino acid levels and the accumulation of lipid droplets. In addition, SCO2 loss resulted in hyperactivation of the insulin-like growth factor 1 receptor (IGF1R)/AKT axis with paradoxical downregulation of mTOR signaling, which was accompanied by increased AMP-activated kinase activity. Accordingly, abrogation of SCO2 expression appears to increase the sensitivity of cells to IGF1R and AKT, but not mTOR inhibitors. Finally, the loss of SCO2 was associated with reduced proliferation and enhanced migration of HCT116 cells. Collectively, herein we describe potential adaptive signaling and metabolic perturbations triggered by mitochondrial complex IV dysfunction.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Chaperonas Moleculares , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células HCT116 , Humanos , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Ribosome profiling (riboseq) has opened the possibilities for the genome-wide studies of translation in all living organisms. This method is based on deep sequencing of mRNA fragments protected by the ribosomes from hydrolysis by ribonucleases, the so-called ribosomal footprints (RFPs). Ribosomal profiling together with RNA sequencing allows not only to identify with a reasonable accuracy translated reading frames in the transcriptome, but also to track changes in gene expression in response to various stimuli. Notably, ribosomal profiling in its classical version has certain limitations. The size of the selected mRNA fragments is 25-35 nts, while RFPs of other sizes are usually omitted from analysis. Also, ribosomal profiling "averages" the data from all ribosomes and does not allow to study specific ribosomal complexes associated with particular translation factors. However, recently developed modifications of ribosomal profiling provide answers to a number of questions. Thus, it has become possible to analyze not only elongating, but also scanning and reinitiating ribosomes, to study events associated with the collision of ribosomes during mRNA translation, to discover new ways of cotranslational assembly of multisubunit protein complexes during translation, and to selectively isolate ribosomal complexes associated with certain protein factors. New data obtained using these modified approaches provide a better understanding of the mechanisms of translation regulation and the functional roles of translational apparatus components.
Assuntos
RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Eucariotos/genética , Eucariotos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
While near-cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here, we describe a rare case of highly efficient near-cognate initiation. A CUG triplet located in the 5' leader of POLG messenger RNA (mRNA) initiates almost as efficiently (â¼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF), which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5' of this CUG governs the ratio between POLG (the catalytic subunit of mitochondrial DNA polymerase) and POLGARF synthesized from a single POLG mRNA. Functional investigation of POLGARF suggests a role in extracellular signaling. While unprocessed POLGARF localizes to the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid cleavage and secretion of a POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved â¼160 million y ago due to a mammalian-wide interspersed repeat (MIR) transposition into the 5' leader sequence of the mammalian POLG gene, which became fixed in placental mammals. This discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution.
Assuntos
Códon de Iniciação/genética , DNA Polimerase gama/genética , Filogenia , Biossíntese de Proteínas/genética , Animais , Sequência de Bases , Proteínas de Transporte/genética , Feminino , Humanos , Proteínas Mitocondriais/genética , Fases de Leitura Aberta/genética , Gravidez , RNA Mensageiro/genética , Fases de Leitura/genéticaRESUMO
Eukaryotic translation initiation involves preinitiation ribosomal complex 5'-to-3' directional probing of mRNA for codons suitable for starting protein synthesis. The recognition of codons as starts depends on the codon identity and on its immediate nucleotide context known as Kozak context. When the context is weak (i.e., nonoptimal), leaky scanning takes place during which a fraction of ribosomes continues the mRNA probing. We explored the relationship between the context of AUG codons annotated as starts of protein-coding sequences and the next AUG codon occurrence. We found that AUG codons downstream from weak starts occur in the same frame more frequently than downstream from strong starts. We suggest that evolutionary selection on in-frame AUGs downstream from weak start codons is driven by the advantage of the reduction of wasteful out-of-frame product synthesis and also by the advantage of producing multiple proteoforms from certain mRNAs. We confirmed translation initiation downstream from weak start codons using ribosome profiling data. We also tested translation of alternative start codons in 10 specific human genes using reporter constructs. In all tested cases, initiation at downstream start codons was more productive than at the annotated ones. In most cases, optimization of Kozak context did not completely abolish downstream initiation, and in the specific example of CMPK1 mRNA, the optimized start remained unproductive. Collectively, our work reveals previously uncharacterized forces shaping the evolution of protein-coding genes and points to the plurality of translation initiation and the existence of sequence features influencing start codon selection, other than Kozak context.
Assuntos
Códon de Iniciação , Evolução Molecular , Iniciação Traducional da Cadeia Peptídica , Sequência de Bases , Sequência Conservada , Humanos , Proteínas/genética , RNA Mensageiro/química , Ribossomos/metabolismoRESUMO
TMA20 (MCT-1), TMA22 (DENR) and TMA64 (eIF2D) are eukaryotic translation factors involved in ribosome recycling and re-initiation. They operate with P-site bound tRNA in post-termination or (re-)initiation translation complexes, thus participating in the removal of 40S ribosomal subunit from mRNA stop codons after termination and controlling translation re-initiation on mRNAs with upstream open reading frames (uORFs), as well as de novo initiation on some specific mRNAs. Here we report ribosomal profiling data of S.cerevisiae strains with individual deletions of TMA20, TMA64 or both TMA20 and TMA64 genes. We provide RNA-Seq and Ribo-Seq data from yeast strains grown in the rich YPD or minimal SD medium. We illustrate our data by plotting differential distribution of ribosomal-bound mRNA fragments throughout uORFs in 5'-untranslated region (5' UTR) of GCN4 mRNA and on mRNA transcripts encoded in MAT locus in the mutant and wild-type strains, thus providing a basis for investigation of the role of these factors in the stress response, mating and sporulation. We also document a shift of transcription start site of the APC4 gene which occurs when the neighboring TMA64 gene is replaced by the standard G418-resistance cassette used for the creation of the Yeast Deletion Library. This shift results in dramatic deregulation of the APC4 gene expression, as revealed by our Ribo-Seq data, which can be probably used to explain strong genetic interactions of TMA64 with genes involved in the cell cycle and mitotic checkpoints. Raw RNA-Seq and Ribo-Seq data as well as all gene counts are available in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE122039 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122039).
RESUMO
Short upstream open reading frames (uORFs) are the most prevalent cis-acting regulatory elements in the mammalian transcriptome which can orchestrate mRNA translation. Apart from being "passive roadblocks" that decrease expression of the main coding regions, particular uORFs can serve as specific sensors for changing conditions, thus regulating translation in response to cell stress. Here we report a novel uORF-based regulatory mechanism that is employed under conditions of hyperosmotic stress by at least two human mRNAs, coding for translation reinitiation/recycling factor eIF2D and E3 ubiquitin ligase MDM2. This novel mode of translational control selectively downregulates their expression and requires as few as one uORF. Using a set of reporter mRNAs and fleeting mRNA transfection (FLERT) technique, we provide evidence that the phenomenon does not rely on delayed reinitiation, altered AUG recognition, ribosome stalling, mRNA destabilization or other known mechanisms. Instead, it is based on events taking place at uORF stop codon or immediately downstream. Functional aspects and implications of the novel regulatory mechanism to cell physiology are discussed.
Assuntos
Códon de Iniciação/metabolismo , Fator de Iniciação 2 em Eucariotos/biossíntese , Fases de Leitura Aberta , Pressão Osmótica , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Códon de Iniciação/genética , Fator de Iniciação 2 em Eucariotos/genética , Células HEK293 , Humanos , Proteínas Proto-Oncogênicas c-mdm2/genética , Estabilidade de RNARESUMO
Translation initiation is the rate-limiting step of protein synthesis that is downregulated during the Integrated Stress Response (ISR). Previously, we demonstrated that most human mRNAs that are resistant to this inhibition possess translated upstream open reading frames (uORFs), and that in some cases a single uORF is sufficient for the resistance. Here we developed a computational model of Initiation Complexes Interference with Elongating Ribosomes (ICIER) to gain insight into the mechanism. We explored the relationship between the flux of scanning ribosomes upstream and downstream of a single uORF depending on uORF features. Paradoxically, our analysis predicts that reducing ribosome flux upstream of certain uORFs increases initiation downstream. The model supports the derepression of downstream translation as a general mechanism of uORF-mediated stress resistance. It predicts that stress resistance can be achieved with long slowly decoded uORFs that do not favor translation reinitiation and that start with initiators of low leakiness.
Assuntos
Modelos Genéticos , Fases de Leitura Aberta , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , Ribossomos/genética , Estresse Fisiológico/genética , Arsenitos/farmacologia , Células HEK293 , Humanos , Fosforilação , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Tunicamicina/farmacologiaRESUMO
In addition to acting as template for protein synthesis, messenger RNA (mRNA) often contains sensory sequence elements that regulate this process. Here we report a new mechanism that limits the number of complete protein molecules that can be synthesized from a single mRNA molecule of the human AMD1 gene encoding adenosylmethionine decarboxylase 1 (AdoMetDC). A small proportion of ribosomes translating AMD1 mRNA stochastically read through the stop codon of the main coding region. These readthrough ribosomes then stall close to the next in-frame stop codon, eventually forming a ribosome queue, the length of which is proportional to the number of AdoMetDC molecules that were synthesized from the same AMD1 mRNA. Once the entire spacer region between the two stop codons is filled with queueing ribosomes, the queue impinges upon the main AMD1 coding region halting its translation. Phylogenetic analysis suggests that this mechanism is highly conserved in vertebrates and existed in their common ancestor. We propose that this mechanism is used to count and limit the number of protein molecules that can be synthesized from a single mRNA template. It could serve to safeguard from dysregulated translation that may occur owing to errors in transcription or mRNA damage.
Assuntos
Adenosilmetionina Descarboxilase/genética , Códon de Terminação/genética , Modelos Genéticos , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Fases de Leitura Aberta/genética , Filogenia , Complexo de Endopeptidases do Proteassoma/metabolismo , Processos Estocásticos , Moldes GenéticosRESUMO
Mitochondrial polarisation is paramount for a variety of cellular functions. Under ischemia, mitochondrial membrane potential (ΔΨm) and proton gradient (ΔpH) are maintained via a reversal of mitochondrial F1Fo ATP synthase (mATPase), which can rapidly deplete ATP and drive cells into energy crisis. We found that under normal conditions in cells with disassembled cytochrome c oxidase complex (COX-deficient HCT116), mATPase maintains ΔΨm at levels only 15-20% lower than in WT cells, and for this utilises relatively little ATP. For a small energy expenditure, mATPase enables mitochondrial ΔpH, protein import, Ca2+ turnover, and supports free radical detoxication machinery enlarged to protect the cells from oxidative damage. Whereas in COX-deficient cells the main source of ATP is glycolysis, the ΔΨm is still maintained upon inhibition of the adenine nucleotide translocators with bongkrekic acid and carboxyatractyloside, indicating that the role of ANTs is redundant, and matrix substrate level phosphorylation alone or in cooperation with ATP-Mg/Pi carriers can continuously support the mATPase activity. Intriguingly, we found that mitochondrial complex III is active, and it contributes not only to free radical production, but also to ΔΨm maintenance and energy budget of COX-deficient cells. Overall, this study demonstrates that F1Fo ATP synthase can support general mitochondrial and cellular functions, working in extremely efficient 'energy saving' reverse mode and flexibly recruiting free radical detoxication and ATP producing / transporting pathways.
Assuntos
Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Metabolismo Energético/genética , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Trifosfato de Adenosina/metabolismo , Carcinoma/genética , Carcinoma/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Deficiência de Citocromo-c Oxidase/genética , Deficiência de Citocromo-c Oxidase/metabolismo , Deficiência de Citocromo-c Oxidase/patologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/patologia , Fosforilação OxidativaRESUMO
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/metabolismoRESUMO
mRNAs lacking 5' untranslated regions (leaderless mRNAs) are molecular relics of an ancient translation initiation pathway. Nevertheless, they still represent a significant portion of transcriptome in some taxons, including a number of eukaryotic species. In bacteria and archaea, the leaderless mRNAs can bind non-dissociated 70 S ribosomes and initiate translation without protein initiation factors involved. Here we use the Fleeting mRNA Transfection technique (FLERT) to show that translation of a leaderless reporter mRNA is resistant to conditions when eIF2 and eIF4F, two key eukaryotic translation initiation factors, are inactivated in mammalian cells. We report an unconventional translation initiation pathway utilized by the leaderless mRNA in vitro, in addition to the previously described 80S-, eIF2-, or eIF2D-mediated modes. This mechanism is a bacterial-like eIF5B/IF2-assisted initiation that has only been reported for hepatitis C virus-like internal ribosome entry sites (IRESs). Therefore, the leaderless mRNA is able to take any of four different translation initiation pathways in eukaryotes.
Assuntos
Células Eucarióticas/fisiologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , RNA Mensageiro/metabolismo , Sistema Livre de Células , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Hepatite C/genética , Humanos , Sítios Internos de Entrada Ribossomal , Complexos Multiproteicos , Biossíntese de Proteínas , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Transfecção/métodosRESUMO
Ribosome profiling (Ribo-seq), a promising technology for exploring ribosome decoding rates, is characterized by the presence of infrequent high peaks in ribosome footprint density and by long alignment gaps. Here, to reduce the impact of data heterogeneity we introduce a simple normalization method, Ribo-seq Unit Step Transformation (RUST). RUST is robust and outperforms other normalization techniques in the presence of heterogeneous noise. We illustrate how RUST can be used for identifying mRNA sequence features that affect ribosome footprint densities globally. We show that a few parameters extracted with RUST are sufficient for predicting experimental densities with high accuracy. Importantly the application of RUST to 30 publicly available Ribo-seq data sets revealed a substantial variation in sequence determinants of ribosome footprint frequencies, questioning the reliability of Ribo-seq as an accurate representation of local ribosome densities without prior quality control. This emphasizes our incomplete understanding of how protocol parameters affect ribosome footprint densities.
Assuntos
RNA Mensageiro/metabolismo , Ribossomos/química , Animais , Códon , Simulação por Computador , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Modelos Estatísticos , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Biossíntese de Proteínas , Reprodutibilidade dos Testes , Ribossomos/ultraestrutura , Leveduras/metabolismoRESUMO
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 5ARESUMO
Protein synthesis in eukaryotes is subject to stringent control. The misregulation of translation of certain mRNAs is often a hallmark of many diseases, including malignancies and autoimmune disorders. To understand why and how it happens, it is important to investigate the translational control of specific mRNAs. In this case, one could use reporter mRNAs in order to identify cis-acting elements responsible for regulation. Here we overview plasmid DNA (pDNA) and mRNA transfections, their pitfalls and limitations, as well as some emerging applications for mRNA transfection.
Assuntos
Mamíferos/metabolismo , Plasmídeos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Genes Reporter , Humanos , Mamíferos/genética , Sequências Reguladoras de Ácido Nucleico , TransfecçãoRESUMO
BACKGROUND: Oxygen and glucose metabolism play pivotal roles in many (patho)physiological conditions. In particular, oxygen and glucose deprivation (OGD) during ischemia and stroke results in extensive tissue injury and cell death. RESULTS: Using time-resolved ribosome profiling, we assess gene expression levels in a neural cell line, PC12, during the first hour of OGD. The most substantial alterations are seen to occur within the first 20 minutes of OGD. While transcription of only 100 genes is significantly altered during one hour of OGD, the translation response affects approximately 3,000 genes. This response involves reprogramming of initiation and elongation rates, as well as the stringency of start codon recognition. Genes involved in oxidative phosphorylation are most affected. Detailed analysis of ribosome profiles reveals salient alterations of ribosome densities on individual mRNAs. The mRNA-specific alterations include increased translation of upstream open reading frames, site-specific ribosome pauses, and production of alternative protein isoforms with amino-terminal extensions. Detailed analysis of ribosomal profiles also reveals six mRNAs with translated ORFs occurring downstream of annotated coding regions and two examples of dual coding mRNAs, where two protein products are translated from the same long segment of mRNA, but in two different frames. CONCLUSIONS: These findings uncover novel regulatory mechanisms of translational response to OGD in mammalian cells that are different from the classical pathways such as hypoxia inducible factor (HIF) signaling, while also revealing sophisticated organization of protein coding information in certain genes.