RESUMO
In addition to the main, protein-coding, open reading frame (mORF), many eukaryotic mRNAs contain upstream ORFs (uORFs) initiated at AUG or near-cognate codons residing 5' of the mORF start site. Whereas translation of uORFs generally represses translation of the mORFs, a subset of uORFs serves as a nexus for regulating translation of the mORF. In this review, we summarize the mechanisms by which uORFs can repress or stimulate mRNA translation, highlight uORF-mediated translational repression involving ribosome queuing, and critically evaluate recently described alternatives to the delayed reinitiation model for uORF-mediated regulation of the GCN4/ATF4 mRNAs.
Assuntos
Biossíntese de Proteínas , Ribossomos , Códon de Iniciação/genética , Códon/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fases de Leitura Aberta/genéticaRESUMO
Polyamines, small organic polycations, are essential for cell viability, and their physiological levels are homeostatically maintained by post-transcriptional regulation of key biosynthetic enzymes. In addition to de novo synthesis, cells can also take up polyamines; however, identifying cellular polyamine transporters has been challenging. Here we show that the S. cerevisiae HOL1 mRNA is under translational control by polyamines, and we reveal that the encoded membrane transporter Hol1 is a high-affinity polyamine transporter and is required for yeast growth under limiting polyamine conditions. Moreover, we show that polyamine inhibition of the translation factor eIF5A impairs translation termination at a Pro-Ser-stop motif in a conserved upstream open reading frame on the HOL1 mRNA to repress Hol1 synthesis under conditions of elevated polyamines. Our findings reveal that polyamine transport, like polyamine biosynthesis, is under translational autoregulation by polyamines in yeast, highlighting the extensive control cells impose on polyamine levels.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Poliaminas/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Fases de Leitura Aberta , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Cells utilize transcriptional and posttranscriptional mechanisms to alter gene expression in response to environmental cues. Gene-specific controls, including changing the translation of specific messenger RNAs (mRNAs), provide a rapid means to respond precisely to different conditions. Upstream open reading frames (uORFs) are known to control the translation of mRNAs. Recent studies in bacteria and eukaryotes have revealed the functions of evolutionarily conserved uORF-encoded peptides. Some of these uORF-encoded nascent peptides enable responses to specific metabolites to modulate the translation of their mRNAs by stalling ribosomes and through ribosome stalling may also modulate the level of their mRNAs. In this review, we highlight several examples of conserved uORF nascent peptides that stall ribosomes to regulate gene expression in response to specific metabolites in bacteria, fungi, mammals, and plants.
Assuntos
Fases de Leitura Aberta/genética , Peptídeos/genética , Biossíntese de Proteínas/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , RNA Mensageiro/genética , Ribossomos/genéticaRESUMO
The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/biossíntese , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Células HEK293 , Antígenos de Histocompatibilidade Classe I/imunologia , Interações Hospedeiro-Patógeno , Humanos , Vigilância Imunológica , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Melanoma/imunologia , Melanoma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCC's upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.
Assuntos
Proteínas de Transporte/biossíntese , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Poliaminas/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Códon de Iniciação , Sequência Conservada , Células HEK293 , Humanos , Camundongos , Fases de Leitura Aberta , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Diphthamide (DPH), a conserved amino acid modification on eukaryotic translation elongation factor eEF2, is synthesized via a complex, multi-enzyme pathway. While DPH is non-essential for cell viability and its function has not been resolved, diphtheria and other bacterial toxins ADP-ribosylate DPH to inhibit translation. Characterizing Saccharomyces cerevisiae mutants that lack DPH or show synthetic growth defects in the absence of DPH, we show that loss of DPH increases resistance to the fungal translation inhibitor sordarin and increases -1 ribosomal frameshifting at non-programmed sites during normal translation elongation and at viral programmed frameshifting sites. Ribosome profiling of yeast and mammalian cells lacking DPH reveals increased ribosomal drop-off during elongation, and removal of out-of-frame stop codons restores ribosomal processivity on the ultralong yeast MDN1 mRNA. Finally, we show that ADP-ribosylation of DPH impairs the productive binding of eEF2 to elongating ribosomes. Our results reveal that loss of DPH impairs the fidelity of translocation during translation elongation resulting in increased rates of ribosomal frameshifting throughout elongation and leading to premature termination at out-of-frame stop codons. We propose that the costly, yet non-essential, DPH modification has been conserved through evolution to maintain translational fidelity despite being a target for inactivation by bacterial toxins.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Fator 2 de Elongação de Peptídeos , Saccharomyces cerevisiae , Animais , Toxinas Bacterianas/metabolismo , Códon de Terminação/metabolismo , Mamíferos/genética , Fator 2 de Elongação de Peptídeos/química , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismoRESUMO
Translation start site selection in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the eukaryotic translation initiation factors eIF1 and eIF5. In a search of mammalian genes, we identified five homeobox (Hox) gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. An analysis of published cap analysis of gene expression sequencing (CAGE-seq) data and generated CAGE-seq data for messenger RNAs (mRNAs) from mouse somites revealed that the 5' leaders of Hox mRNAs of interest contain conserved uORFs, are generally much shorter than reported, and lack previously proposed internal ribosome entry site elements. We show that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of Hox reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin leads to lower stringency of start codon selection. Thus, altering global translation can confer gene-specific effects through altered start codon selection stringency.
Assuntos
Códon de Iniciação , Evolução Molecular , Genes Homeobox , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Camundongos , Fases de Leitura AbertaRESUMO
Polyamines are organic polycations that bind to a variety of cellular molecules, including nucleic acids. Within cells, polyamines contribute to both the efficiency and fidelity of protein synthesis. In addition to directly acting on the translation apparatus to stimulate protein synthesis, the polyamine spermidine serves as a precursor for the essential post-translational modification of the eukaryotic translation factor 5A (eIF5A), which is required for synthesis of proteins containing problematic amino acid sequence motifs, including polyproline tracts, and for termination of translation. The impact of polyamines on translation is highlighted by autoregulation of the translation of mRNAs encoding key metabolic and regulatory proteins in the polyamine biosynthesis pathway, including S-adenosylmethionine decarboxylase (AdoMetDC), antizyme (OAZ), and antizyme inhibitor 1 (AZIN1). Here, we highlight the roles of polyamines in general translation and also in the translational regulation of polyamine biosynthesis.
Assuntos
Poliaminas/metabolismo , Biossíntese de Proteínas , Proteínas/genética , Animais , Humanos , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Although stop codon readthrough is used extensively by viruses to expand their gene expression, verified instances of mammalian readthrough have only recently been uncovered by systems biology and comparative genomics approaches. Previously, our analysis of conserved protein coding signatures that extend beyond annotated stop codons predicted stop codon readthrough of several mammalian genes, all of which have been validated experimentally. Four mRNAs display highly efficient stop codon readthrough, and these mRNAs have a UGA stop codon immediately followed by CUAG (UGA_CUAG) that is conserved throughout vertebrates. Extending on the identification of this readthrough motif, we here investigated stop codon readthrough, using tissue culture reporter assays, for all previously untested human genes containing UGA_CUAG. The readthrough efficiency of the annotated stop codon for the sequence encoding vitamin D receptor (VDR) was 6.7%. It was the highest of those tested but all showed notable levels of readthrough. The VDR is a member of the nuclear receptor superfamily of ligand-inducible transcription factors, and it binds its major ligand, calcitriol, via its C-terminal ligand-binding domain. Readthrough of the annotated VDR mRNA results in a 67 amino acid-long C-terminal extension that generates a VDR proteoform named VDRx. VDRx may form homodimers and heterodimers with VDR but, compared with VDR, VDRx displayed a reduced transcriptional response to calcitriol even in the presence of its partner retinoid X receptor.
Assuntos
Calcitriol/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Códon de Terminação , Regulação da Expressão Gênica/efeitos dos fármacos , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Receptores de Calcitriol/genética , Células HEK293 , Células HeLa , Humanos , Fases de Leitura Aberta , RNA Mensageiro/genética , Receptores de Calcitriol/biossínteseRESUMO
Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systematic mutagenesis approach. One gene, OPRL1, encoding an opiate receptor, displayed extremely efficient levels of readthrough (â¼31%) in HEK-293T cells. Signals both 5' and 3' of the OPRL1 stop codon contribute to this high level of readthrough. The sequence UGA-CUA alone can support 1.5% readthrough, underlying its importance.
Assuntos
Códon de Terminação , Biossíntese de Proteínas , Aminoglicosídeos/farmacologia , Animais , Antibacterianos/farmacologia , Aquaporina 4/genética , Sequência Conservada , Células HEK293 , Humanos , Proteína Quinase 10 Ativada por Mitógeno/genética , Motivos de Nucleotídeos , Filogenia , Biossíntese de Proteínas/efeitos dos fármacos , Receptores Opioides/genética , Receptores Opioides kappa/genética , Receptor de NociceptinaRESUMO
In eukaryotic cells initiation may occur from near-cognate codons that differ from AUG by a single nucleotide. The stringency of start codon selection impacts the efficiency of initiation at near-cognate codons and the efficiency of initiation at AUG codons in different contexts. We used a codon-optimized firefly luciferase reporter initiated with AUG or each of the nine near-cognate codons in preferred context to examine the stringency of start codon selection in the model filamentous fungus Neurospora crassa. In vivo results indicated that the hierarchy of initiation at start codons in N. crassa (AUG â« CUG > GUG > ACG > AUA ≈ UUG > AUU > AUC) is similar to that in human cells. Similar results were obtained by translating mRNAs in a homologous N. crassa in vitro translation system or in rabbit reticulocyte lysate. We next examined the efficiency of initiation at AUG, CUG, and UUG codons in different contexts in vitro. The preferred context was more important for efficient initiation from near-cognate codons than from AUG. These studies demonstrated that near-cognate codons are used for initiation in N. crassa. Such events could provide additional coding capacity or have regulatory functions. Analyses of the 5'-leader regions in the N. crassa transcriptome revealed examples of highly conserved near-cognate codons in preferred contexts that could extend the N termini of the predicted polypeptides.
Assuntos
Códon de Iniciação , Neurospora crassa/genética , Neurospora crassa/metabolismo , Sequência de Aminoácidos , Sistema Livre de Células , Códon , Biologia Computacional/métodos , DNA Complementar/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Peptídeos/química , Plasmídeos/metabolismo , Prevalência , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Ca(2+) signaling is essential for bone homeostasis and skeletal development. Here, we show that the transient receptor potential canonical 1 (TRPC1) channel and the inhibitor of MyoD family, I-mfa, function antagonistically in the regulation of osteoclastogenesis. I-mfa null mice have an osteopenic phenotype characterized by increased osteoclast numbers and surface, which are normalized in mice lacking both Trpc1 and I-mfa. In vitro differentiation of pre-osteoclasts derived from I-mfa-deficient mice leads to an increased number of mature osteoclasts and higher bone resorption per osteoclast. These parameters return to normal levels in osteoclasts derived from double mutant mice. Consistently, whole cell currents activated in response to the depletion of intracellular Ca(2+) stores are larger in pre-osteoclasts derived from I-mfa knock-out mice compared with currents in wild type mice and normalized in cells derived from double mutant mice, suggesting a cell-autonomous effect of I-mfa on TRPC1 in these cells. A new splice variant of TRPC1 (TRPC1ε) was identified in early pre-osteoclasts. Heterologous expression of TRPC1ε in HEK293 cells revealed that it is unique among all known TRPC1 isoforms in its ability to amplify the activity of the Ca(2+) release-activated Ca(2+) (CRAC) channel, mediating store-operated currents. TRPC1ε physically interacts with Orai1, the pore-forming subunit of the CRAC channel, and I-mfa is recruited to the TRPC1ε-Orai1 complex through TRPC1ε suppressing CRAC channel activity. We propose that the positive and negative modulation of the CRAC channel by TRPC1ε and I-mfa, respectively, fine-tunes the dynamic range of the CRAC channel regulating osteoclastogenesis.
Assuntos
Osteoclastos/citologia , Canais de Cátion TRPC/fisiologia , Animais , Sequência de Bases , Divisão Celular , Linhagem Celular , Códon , Primers do DNA , Humanos , Camundongos , Camundongos Knockout , Biossíntese de Proteínas , RNA Mensageiro/genética , Canais de Cátion TRPC/genéticaRESUMO
An AUG in an optimal nucleotide context is the preferred translation initiation site in eukaryotic cells. Interactions among translation initiation factors, including eIF1 and eIF5, govern start codon selection. Experiments described here showed that high intracellular eIF5 levels reduced the stringency of start codon selection in human cells. In contrast, high intracellular eIF1 levels increased stringency. High levels of eIF5 induced translation of inhibitory upstream open reading frames (uORFs) in eIF5 mRNA that initiate with AUG codons in conserved poor contexts. This resulted in reduced translation from the downstream eIF5 start codon, indicating that eIF5 autoregulates its own synthesis. As with eIF1, which is also autoregulated through translation initiation, features contributing to eIF5 autoregulation show deep evolutionary conservation. The results obtained provide the basis for a model in which auto- and cross-regulation of eIF5 and eIF1 translation establish a regulatory feedback loop that would stabilize the stringency of start codon selection.
Assuntos
Códon de Iniciação , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Sequência Conservada , Fator de Iniciação 1 em Eucariotos/metabolismo , Células HEK293 , Homeostase , Humanos , Fatores de Iniciação de Peptídeos/biossíntese , Fatores de Iniciação de Peptídeos/genética , RNA Mensageiro/química , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Translation initiation defines the identity of a synthesized protein through selection of a translation start site on a messenger RNA. This process is essential to well-controlled protein synthesis, modulated by stress responses, and dysregulated in many human diseases. The eukaryotic initiation factors eIF1 and eIF5 interact with the initiator methionyl-tRNAi Met on the 40S ribosomal subunit to coordinate start site selection. Here, using single-molecule analysis of in vitro reconstituted human initiation combined with translation assays in cells, we examine eIF1 and eIF5 function. During translation initiation on a panel of RNAs, we monitored both proteins directly and in real time using single-molecule fluorescence. As expected, eIF1 loaded onto mRNAs as a component of the 43S initiation complex. Rapid (~ 2 s) eIF1 departure required a translation start site and was delayed by alternative start sites and a longer 5' untranslated region (5'UTR). After its initial departure, eIF1 rapidly and transiently sampled initiation complexes, with more prolonged sampling events on alternative start sites. By contrast, eIF5 only transiently bound initiation complexes late in initiation immediately prior to association of eIF5B, which allowed joining of the 60S ribosomal subunit. eIF5 association required the presence of a translation start site and was inhibited and destabilized by alternative start sites. Using both knockdown and overexpression experiments in human cells, we validated that eIF1 and eIF5 have opposing roles during initiation. Collectively, our findings demonstrate how multiple eIF1 and eIF5 binding events control start-site selection fidelity throughout initiation, which is tuned in response to changes in the levels of both proteins.
RESUMO
We have conducted a study on the nonlinear absorption properties of nitrogen-vacancy color centers in processed nanodiamonds. Their two-photon (2P) spectra disclose distinguishable features for the two charge states in which the center exists. The 2P absorption cross section is found to be between 0.1 and 0.5 GM in the wavelength range between 800 and 1040 nm. In addition, the center demonstrates the feature of strong 2P absorption for its neutral charge state below 1000 nm excitation wavelength and predominant 2P absorption by the negative charge state above this wavelength.
RESUMO
In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5' cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized--both for increased coding capacity and potentially also for novel regulatory mechanisms--remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5' untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data.
Assuntos
Códon de Iniciação/química , Evolução Molecular , Regiões 5' não Traduzidas , Processamento Alternativo , Sequência de Bases , Western Blotting , Sequência Conservada , Humanos , Filogenia , RNA Mensageiro/química , Alinhamento de Sequência , Análise de Sequência de RNARESUMO
The central feature of standard eukaryotic translation initiation is small ribosome subunit loading at the 5' cap followed by its 5' to 3' scanning for a start codon. The preferred start is an AUG codon in an optimal context. Elaborate cellular machinery exists to ensure the fidelity of start codon selection. Eukaryotic initiation factor 1 (eIF1) plays a central role in this process. Here we show that the translation of eIF1 homologs in eukaryotes from diverse taxa involves initiation from an AUG codon in a poor context. Using human eIF1 as a model, we show that this poor context is necessary for an autoregulatory negative feedback loop in which a high level of eIF1 inhibits its own translation, establishing that variability in the stringency of start codon selection is used for gene regulation in eukaryotes. We show that the stringency of start codon selection (preferential utilization of optimal start sites) is increased to a surprising degree by overexpressing eIF1. The capacity for the cellular level of eIF1 to impact initiation through the variable stringency of initiation codon selection likely has significant consequences for the proteome in eukaryotes.
Assuntos
Códon de Iniciação , Fator de Iniciação 1 em Eucariotos/fisiologia , Linhagem Celular , Humanos , TransfecçãoRESUMO
In many eukaryotic mRNAs one or more short 'upstream' open reading frames, uORFs, precede the initiator of the main coding sequence. Upstream ORFs are functionally diverse as illustrated by their variety of features in polyamine pathway biosynthetic mRNAs. Their propensity to act as sensors for regulatory circuits and to amplify the signals likely explains their occurrence in most polyamine pathway mRNAs. The uORF-mediated polyamine responsive autoregulatory circuits found in polyamine pathway mRNAs exemplify the translationally regulated dynamic interface between components of the proteome and metabolism.
Assuntos
Regulação da Expressão Gênica , Fases de Leitura Aberta , Poliaminas/metabolismo , Biossíntese de Proteínas , Acetiltransferases/genética , Acetiltransferases/metabolismo , Adenosilmetionina Descarboxilase/genética , Adenosilmetionina Descarboxilase/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico , Proteínas/genética , Proteínas/metabolismo , Espermina Sintase/genética , Espermina Sintase/metabolismoRESUMO
The 5' regions of eukaryotic mRNAs often contain upstream open reading frames (uORFs). The Neurospora crassa arg-2 uORF encodes the 24-residue arginine attenuator peptide (AAP). This regulatory uORF-encoded peptide, which is evolutionarily conserved in fungal transcripts specifying an arginine biosynthetic enzyme, functions as a nascent peptide within the ribosomal tunnel and negatively regulates gene expression. The nascent AAP causes ribosomes to stall at the uORF stop codon in response to arginine, thus, blocking ribosomes from reaching the ARG-2 initiation codon. Here scanning mutagenesis with alanine and proline was performed to systematically determine which AAP residues were important for conferring regulation. Changing many of the most highly conserved residues (Asp-12, Tyr-13, Lys-14, and Trp-19) abolished regulatory function. The minimal functional domain of the AAP was determined by positioning AAP sequences internally within a large polypeptide. Pulse-chase analyses revealed that residues 9-20 of the AAP composed the minimal domain that was sufficient to confer regulatory function. An extensive analysis of predicted fungal AAPs revealed that the minimal functional domain of the N. crassa AAP corresponded closely to the region that was most highly conserved among the fungi. We also observed that the tripeptide RGD could function similarly to arginine in triggering AAP-mediated ribosome stalling. These studies provide a better understanding of the elements required for a nascent peptide and a small regulatory molecule to control translational processes.
Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Fragmentos de Peptídeos/metabolismo , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Substituição de Aminoácidos , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Códon de Iniciação/genética , Códon de Iniciação/metabolismo , Proteínas Fúngicas/genética , Mutação de Sentido Incorreto , Neurospora crassa/genética , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , RNA Fúngico/genética , RNA Fúngico/metabolismo , Ribossomos/genéticaRESUMO
In a minority of eukaryotic mRNAs, a small functional upstream ORF (uORF), often performing a regulatory role, precedes the translation start site for the main product(s). Here, conserved uORFs in numerous ornithine decarboxylase homologs are identified from yeast to mammals. Most have noncanonical evolutionarily conserved start codons, the main one being AUU, which has not been known as an initiator for eukaryotic chromosomal genes. The AUG-less uORF present in mouse antizyme inhibitor, one of the ornithine decarboxylase homologs in mammals, mediates polyamine-induced repression of the downstream main ORF. This repression is part of an autoregulatory circuit, and one of its sensors is the AUU codon, which suggests that translation initiation codon identity is likely used for regulation in eukaryotes.