RESUMO
Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings.
Assuntos
DNA-Formamidopirimidina Glicosilase/química , Sondas Moleculares/química , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , Recombinases/química , DNA-Formamidopirimidina Glicosilase/metabolismo , Escherichia coli O157/genética , Humanos , Sondas Moleculares/metabolismo , RNA Ribossômico 16S/genética , Recombinases/metabolismoRESUMO
Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.
Assuntos
Códon de Terminação/genética , Vírus da Febre do Carrapato do Colorado/genética , Terminação Traducional da Cadeia Peptídica/genética , RNA/genética , Animais , Sequência de Bases , Sistema Livre de Células , Códon de Terminação/metabolismo , Vírus da Febre do Carrapato do Colorado/metabolismo , Dermacentor/genética , Dermacentor/metabolismo , Insetos/genética , Insetos/metabolismo , Dados de Sequência Molecular , Mutação , Biossíntese de Proteínas/genética , RNA/metabolismo , Ribossomos/metabolismoRESUMO
Termination-dependent reinitiation is used to co-ordinately regulate expression of the M1 and BM2 open-reading frames (ORFs) of the dicistronic influenza B segment 7 RNA. The start codon of the BM2 ORF overlaps the stop codon of the M1 ORF in the pentanucleotide UAAUG and â¼10% of ribosomes terminating at the M1 stop codon reinitiate translation at the overlapping AUG. BM2 synthesis requires the presence of, and translation through, 45 nt of RNA immediately upstream of the UAAUG, known as the 'termination upstream ribosome binding site' (TURBS). This region may tether ribosomal 40S subunits to the mRNA following termination and a short region of the TURBS, motif 1, with complementarity to helix 26 of 18S rRNA has been implicated in this process. Here, we provide further evidence for a direct interaction between mRNA and rRNA using antisense oligonucleotide targeting and functional analysis in yeast cells. The TURBS also binds initiation factor eIF3 and we show here that this protein stimulates reinitiation from both wild-type and defective TURBS when added exogenously, perhaps by stabilising ribosome-mRNA interactions. Further, we show that the position of the TURBS with respect to the UAAUG overlap is crucial, and that termination too far downstream of the 18S complementary sequence inhibits the process, probably due to reduced 40S tethering. However, in reporter mRNAs where the restart codon alone is moved downstream, termination-reinitiation is inhibited but not abolished, thus the site of reinitiation is somewhat flexible. Reinitiation on distant AUGs is not inhibited in eIF4G-depleted RRL, suggesting that the tethered 40S subunit can move some distance without a requirement for linear scanning.
Assuntos
Genoma Viral/genética , Vírus da Influenza B/genética , Iniciação Traducional da Cadeia Peptídica/genética , Terminação Traducional da Cadeia Peptídica/genética , RNA Viral/genética , Animais , Sequência de Bases , Sítios de Ligação , Códon de Iniciação/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Camundongos , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Oligonucleotídeos Antissenso/genética , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Coelhos , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/citologiaRESUMO
Viruses utilize a number of translational control mechanisms to regulate the relative expression levels of viral proteins on polycistronic mRNAs. One such mechanism, that of termination-dependent reinitiation, has been described in a number of both negative- and positive-strand RNA viruses. Dicistronic RNAs which exhibit termination-reinitiation typically have a start codon of the 3'-ORF (open reading frame) proximal to the stop codon of the upstream ORF. For example, the segment 7 RNA of influenza B is dicistronic, and the stop codon of the M1 ORF and the start codon of the BM2 ORF overlap in the pentanucleotide UAAUG (the stop codon of M1 is shown in bold and the start codon of BM2 is underlined). Recent evidence has highlighted the potential importance of mRNA-rRNA interactions in reinitiation on caliciviral and influenza B viral RNAs, probably used to tether 40S ribosomal subunits to the RNA after termination in time for initiation factors to be recruited to the AUG of the downstream ORF. The present review summarizes how such interactions regulate reinitiation in an array of RNA viruses, and discusses what is known about reinitiation in viruses that do not rely on apparent mRNA-rRNA interactions.
Assuntos
Iniciação Traducional da Cadeia Peptídica/genética , Terminação Traducional da Cadeia Peptídica/genética , Vírus de RNA/genética , Vírus de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Animais , Sequência de Bases , Códon de Iniciação , Códon de Terminação , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Viral/química , Alinhamento de Sequência , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
BACKGROUND: Expression of the minor virion structural protein VP2 of the calicivirus murine norovirus (MNV) is believed to occur by the unusual mechanism of termination codon-dependent reinitiation of translation. In this process, following translation of an upstream open reading frame (ORF) and termination at the stop codon, a proportion of 40S subunits remain associated with the mRNA and reinitiate at the AUG of a downstream ORF, which is typically in close proximity. Consistent with this, the VP2 start codon (AUG) of MNV overlaps the stop codon of the upstream VP1 ORF (UAA) in the pentanucleotide UAAUG. PRINCIPAL FINDINGS: Here, we confirm that MNV VP2 expression is regulated by termination-reinitiation and define the mRNA sequence requirements. Efficient reintiation is dependent upon 43 nt of RNA immediately upstream of the UAAUG site. Chemical and enzymatic probing revealed that the RNA in this region is not highly structured and includes an essential stretch of bases complementary to 18S rRNA helix 26 (Motif 1). The relative position of Motif 1 with respect to the UAAUG site impacts upon the efficiency of the process. Termination-reinitiation in MNV was also found to be relatively insensitive to the initiation inhibitor edeine. CONCLUSIONS: The termination-reinitiation signal of MNV most closely resembles that of influenza BM2. Similar to other viruses that use this strategy, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the VP1 stop codon. Our data also indicate that accurate recognition of the VP2 ORF AUG is not a pre-requisite for efficient reinitiation of translation in this system.
Assuntos
Norovirus/metabolismo , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Proteínas Virais/metabolismo , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Códon de Iniciação/genética , Códon de Terminação/genética , Edeína/farmacologia , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Norovirus/efeitos dos fármacos , Conformação de Ácido Nucleico , Nucleotídeos/genética , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , RNA Complementar/genética , RNA Mensageiro/genética , RNA Ribossômico 18S/genética , RNA Viral/química , RNA Viral/genética , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression. Termination-reinitiation is dependent upon 45 nucleotide (nt) of RNA immediately upstream of the UAAUG pentanucleotide, which includes an essential stretch complementary to 18S rRNA helix 26. Thus, similar to the caliciviruses, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the M1 stop codon. Consistent with this, repositioning of the M1 stop codon more than 24 nt downstream from the BM2 start codon inhibited BM2 expression. RNA structure probing revealed that the RNA upstream of the UAAUG overlap is not highly structured, but upon encountering the M1 stop codon by the ribosome, a stem-loop may form immediately 5' of the ribosome, with the 18S rRNA complementary region in the apical loop and in close proximity to helix 26. Mutational analysis reveals that the normal requirements for start site selection in BM2 expression are suspended, with little effect of initiation codon context and efficient use of noncanonical initiation codons. This suggests that the full complement of initiation factors is not required for the reinitiation process.
Assuntos
Vírus da Influenza B/metabolismo , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Proteínas Virais/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Códon de Iniciação/genética , Códon de Iniciação/metabolismo , Códon de Terminação/genética , Códon de Terminação/metabolismo , Vírus da Influenza B/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica/genética , Terminação Traducional da Cadeia Peptídica/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/genéticaRESUMO
Viruses have evolved a number of translational control mechanisms to regulate the levels of expression of viral proteins on polycistronic mRNAs, including programmed ribosomal frameshifting and stop codon readthrough. More recently, another unusual mechanism has been described, that of termination-dependent re-initiation (also known as stop-start). Here, the AUG start codon of a 3' ORF (open reading frame) is proximal to the termination codon of a uORF (upstream ORF), and expression of the two ORFs is coupled. For example, segment 7 mRNA of influenza B is bicistronic, and the stop codon of the M1 ORF and the start codon of the BM2 ORF overlap in the pentanucleotide UAAUG (stop codon of M1 is shown in boldface and start codon of BM2 is underlined). This short review aims to provide some insights into how this translational coupling process is regulated within different viral systems and to highlight some of the differences in the mechanism of re-initiation on prokaryotic, eukaryotic and viral mRNAs.
Assuntos
Regulação Viral da Expressão Gênica/genética , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Animais , Códon de Terminação/genética , Humanos , RNA Ribossômico 18S/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In pancreatic beta-cells, glucose causes a rapid increase in the rate of protein synthesis. However, the mechanism by which this occurs is poorly understood. In this report, we demonstrate, in the pancreatic beta-cell line MIN6, that glucose stimulates the recruitment of ribosomes onto the mRNA, indicative of an increase in the rate of the initiation step of protein synthesis. This increase in the rate of initiation is not mediated through an increase in the availability of the initiation complex eIF4F, because glucose is unable to stimulate eIF4F assembly or, in the absence of amino acids, modulate the phosphorylation status of 4E-BP1. Moreover, in MIN6 cells and isolated islets of Langerhans, rapamycin, an inhibitor of the mammalian target of rapamycin, only partially inhibited glucose-stimulated protein synthesis. However, we show that glucose stimulates the dephosphorylation of eIF2 alpha in MIN6 cells and the assembly of the translational ternary complex, eIF2-GTP.Met-tRNAi, in both MIN6 cells and islets of Langerhans. The changes in the phosphorylation of eIF2 alpha are not mediated by the PKR-like endoplasmic reticulum eIF2 alpha kinase (PERK), because PERK is not phosphorylated at low glucose concentrations and overexpression of a dominant negative form of PERK has no significant effect on either glucose-stimulated protein synthesis or the phosphorylation of eIF2 alpha. Taken together, these results indicate that glucose-stimulated protein synthesis in pancreatic beta-cells is regulated by a mechanism largely independent of the activity of mammalian target of rapamycin, but which is likely to be dependent on the availability of the translational ternary complex, regulated by the phosphorylation status of eIF2 alpha.
