Prevalence and distribution of hepatitis C virus genotypes in Spain during the 2000-2015 period (the GEHEP 005 study).

We report the largest study on the prevalence and distribution of HCV genotypes in Spain (2000-2015), and we relate them with clinical, epidemiological and virological factors. Patients from 29 hospitals in 10 autonomous communities (Andalusia, Aragon, Castilla-Leon, Catalonia, Galicia, Canary Islands, Madrid Community, Valencian Community, Murcia Region and Basque Country) have been studied. Annual distribution of HCV genotypes and subtypes, as well as gender, age, transmission route, HIV and/or HBV coinfection, and treatment details were recorded. We included 48595 chronically HCV-infected patients with the following characteristics: median age 51 years (IQR, 44-58), 67.9% male, 19.1% HIV-coinfected, 23.5% HBV-coinfected. Parenteral transmission route was the most frequent (58.7%). Genotype distribution was 66.9% GT1 (24.9% subtype 1a and 37.9% subtype 1b), 2.8% GT2, 17.3% GT3, 11.4% GT4 and 0.1% GT5 and 0.02% GT6. LiPA was the most widely HCV genotyping test used (52.4%). HCV subtype 1a and genotypes 3 and 4 were closely associated with male gender, parenteral route of infection and HIV and HBV coinfection; in contrast, subtype 1b and genotype 2 were associated with female gender, nonparenteral route and mono-infection. Age was related to genotype distribution, and different patterns of distribution and biodiversity index were observed between different geographical areas. Finally, we describe how treatment and changes in transmission routes may have affected HCV genotype prevalence and distribution patterns. We present the most recent data on molecular epidemiology of hepatitis C virus in Spain. This study confirms that genotype distributions vary with age, sex, HIV and HBV coinfection and within geographical areas and epidemiological groups.

Mid- to long-term results of SuPerLap single-port treatment in inguinal hernia. / Resultados a medio-largo plazo del tratamiento monopuerto SuPerLap de la hernia inguinal.

INTRODUCTION: Laparoscopic treatment of inguinal hernia is gaining popularity in many hospitals, but the use of working channel scopes is not as widely extended. We present our long-term experience with the SuPerLap (laparoscopic-assisted percutaneous suture) technique described by Rosell et al.(1) for epigastric hernia repair in the percutaneous, single-port treatment of inguinal hernia using working channel scopes. MATERIALS AND METHODS: A retrospective analysis of a series of male patients with congenital inguinal hernia undergoing surgery from February 2017 to December 2020 was carried out. A 5 mm-0º pleuroscope with a 3.5 mm working channel, a 20 G epidural needle, a 36 cm/3.5 mm laparoscopic Maryland dissector, and 3-0 polypropylene and polyester sutures were used. RESULTS: 384 inguinal hernia repairs using the SuPerLap technique were performed in 295 male patients - 206 unilateral repairs and 89 bilateral repairs. In 24 bilateral cases (26.95%), preoperative diagnosis had been unilateral. Mean age was two years (2 weeks-13 years). Mean operating time was 14 minutes (6-50 min) for unilateral repair, and 27 minutes (14-80 min) for bilateral repair. There were two cases of epigastric vessel damage, and one case of early recurrence in a newborn, who successfully underwent re-intervention using the SuPerLap technique. No late complications were recorded after a mean follow-up of 1-36 months. CONCLUSIONS: Working channel scopes using the SuPerLap technique avoid additional ports in inguinal hernia repair. They allow for excellent functional results, without visible scars, and minimize spermatic cord manipulation. Laparoscopy allows previously undiagnosed defects to be concomitantly treated.

INTRODUCCION: La laparoscopia en el tratamiento de la hernia inguinal está cada vez más presente en muchos hospitales. El uso de ópticas con canal de trabajo no está tan extendido. Se presenta la experiencia a largo plazo en la aplicación de la técnica SuPerLap (sutura percutánea laparoasistida) propuesta por Rosell y cols.(1) para la reparación de hernias epigástricas en el tratamiento monopuerto, percutáneo de las hernias inguinales mediante el uso de ópticas con canal de trabajo. MATERIAL Y METODO: Serie quirúrgica de hernia inguinal congénita en varones (febrero de 2017-diciembre de 2020). Se utilizó: pleuroscopio de 5 mm-0º con canal de trabajo de 3,5 mm; aguja epidural 20 G; suturas de polipropileno y poliéster 3/0; disector Maryland laparoscópico (36 cm-3,5 mm). RESULTADOS: Se realizaron 384 herniorrafias inguinales según técnica SuPerLap en 295 varones (206 unilaterales, 89 bilaterales). En 24 casos bilaterales (26,95%) el diagnóstico preoperatorio fue unilateral. La edad media fue de dos años (2 semanas-13 años). El tiempo medio quirúrgico fue 14 minutos (6-50 min) en unilaterales, 27 (14-80 min) en bilaterales. Hubo dos casos de lesión de vasos epigástricos y una recidiva precoz en un neonato, reintervenido satisfactoriamente mediante técnica SuPerLap. En un seguimiento de 1-36 meses no hubo complicaciones tardías. CONCLUSIONES: El uso de ópticas con canal de trabajo según técnica SuPerLap posibilita prescindir de puertos adicionales en el tratamiento de la hernia inguinal. Permite resultados funcionales comparables y cirugía sin cicatrices visibles. Minimiza la manipulación del cordón espermático. La laparoscopia permite el tratamiento concomitante de defectos no diagnosticados previamente.

Self-Compacting Recycled Concrete Using Biomass Bottom Ash.

In recent years, the use of self-compacting concrete has been a great advantage and garnered undoubted interest in construction. Due to the environmental impact caused by the consumption of natural aggregates in the manufacture of concrete, a more sustainable approach is needed. An approach for more sustainable construction is to use industrial waste such as bottom ash from the combustion of biomass as a replacement for natural aggregates. This research aims to use biomass bottom ash as a replacement for natural sand (10%, 20% and 30% replacement); in addition, by utilizing a crushing process of the bottom ash, the ash has been used as a filler replacement (replacement 20%, 40% and 60%). The fresh and hardened properties have been evaluated according to the standard. The results show the feasibility of using biomass bottom ash in self-compacting concrete, providing a sustainable alternative in order to minimise environmental impacts related to the extraction and depletion of natural resources.

Making sense of nonsense in yeast.

Messenger RNA (mRNA) degradation is a process that plays an important role in the regulation of gene expression and can be linked to translation. Study of the nonsense-mediated mRNA decay pathway has greatly aided our understanding of the link between these processes. Evidence indicates that this pathway regulates the abundance of both aberrant and wild-type transcripts. Factors involved in this pathway have been identified and recent results indicate that they might also be involved in modulating translation. Here, we discuss the mechanism of nonsense-mediated mRNA decay in the yeast Saccharomyces cerevisiae and the potential role that this pathway can have on the regulation of gene expression.

Replication and control of circular bacterial plasmids.

An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3'-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can "sense" and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.

Characterization of cis-acting sequences and decay intermediates involved in nonsense-mediated mRNA turnover.

Several lines of evidence indicate that the processes of mRNA turnover and translation are intimately linked and that understanding this relationship is critical to elucidating the mechanism of mRNA decay. One clear example of this relationship is the observation that nonsense mutations can accelerate the decay of mRNAs in a process that we term nonsense-mediated mRNA decay. The experiments described here demonstrate that in the yeast Saccharomyces cerevisiae premature translational termination within the initial two-thirds of the PGK1 coding region accelerates decay of that transcript regardless of which of the stop codons is used. Nonsense mutations within the last quarter of the coding region have no effect on PGK1 mRNA decay. The sequences required for nonsense-mediated mRNA decay include a termination codon and specific sequences 3' to the nonsense mutation. Translation of two-thirds of the PGK1 coding region inactivates the nonsense-mediated mRNA decay pathway. This observation explains why carboxyl-terminal nonsense mutations are resistant to accelerated decay. Characterization of the decay of nonsense-containing HIS4 transcripts yielded results mirroring those described above, suggesting that the sequence requirements described for the PGK1 transcript are likely to be a general characteristic of this decay pathway. In addition, an analysis of the decay intermediates of nonsense-containing mRNAs indicates that nonsense-mediated mRNA decay flows through a pathway similar to that described for a class of wild-type transcripts. The initial cleavage event occurs near the 5' terminus of the nonsense-containing transcript and is followed by 5'-->3' exonucleolytic digestion. A model for nonsense-mediated mRNA decay based on these results is discussed.

Identification and characterization of a sequence motif involved in nonsense-mediated mRNA decay.

In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate or reduce the abundance of the mRNA transcribed from that gene, and we call this process nonsense-mediated mRNA decay. We have been investigating the cis-acting sequences involved in this decay pathway. Previous experiments have demonstrated that, in addition to a nonsense codon, specific sequences 3' of a nonsense mutation, which have been defined as downstream elements, are required for mRNA destabilization. The results presented here identify a sequence motif (TGYYGATGYYYYY, where Y stands for either T or C) that can predict regions in genes that, when positioned 3' of a nonsense codon, promote rapid decay of its mRNA. Sequences harboring two copies of the motif from five regions in the PGK1, ADE3, and HIS4 genes were able to function as downstream elements. In addition, four copies of this motif can function as an independent downstream element. The sequences flanking the motif played a more significant role in modulating its activity when fewer copies of the sequence motif were present. Our results indicate the sequences 5' of the motif can modulate its activity by maintaining a certain distance between the sequence motif and the termination codon. We also suggest that the sequences 3' of the motif modulate the activity of the downstream element by forming RNA secondary structures. Consistent with this view, a stem-loop structure positioned 3' of the sequence motif can enhance the activity of the downstream element. This sequence motif is one of the few elements that have been identified that can predict regions in genes that can be involved in mRNA turnover. The role of these sequences in mRNA decay is discussed.

Resultados a medio-largo plazo del tratamiento monopuerto SuPerLap de la hernia inguinal / Mid- to long-term results of SuPerLap single-port treatment in inguinal hernia

Objetivos: La laparoscopia en el tratamiento de la hernia inguinalestá cada vez más presente en muchos hospitales. El uso de ópticas concanal de trabajo no está tan extendido. Se presenta la experiencia a largoplazo en la aplicación de la técnica SuPerLap (sutura percutánea lapa-roasistida) propuesta por Rosell y cols.(1) para la reparación de herniasepigástricas en el tratamiento monopuerto, percutáneo de las herniasinguinales mediante el uso de ópticas con canal de trabajo.Material y método: Serie quirúrgica de hernia inguinal congénitaen varones (febrero de 2017-diciembre de 2020). Se utilizó: pleuroscopiode 5 mm-0º con canal de trabajo de 3,5 mm; aguja epidural 20 G; suturasde polipropileno y poliéster 3/0; disector Maryland laparoscópico (36cm-3,5 mm).Resultados: Se realizaron 384 herniorrafias inguinales según técnicaSuPerLap en 295 varones (206 unilaterales, 89 bilaterales). En 24 casosbilaterales (26,95%) el diagnóstico preoperatorio fue unilateral. La edadmedia fue de dos años (2 semanas-13 años). El tiempo medio quirúrgicofue 14 minutos (6-50 min) en unilaterales, 27 (14-80 min) en bilaterales.Hubo dos casos de lesión de vasos epigástricos y una recidiva precoz enun neonato, reintervenido satisfactoriamente mediante técnica SuPer-Lap. En un seguimiento de 1-36 meses no hubo complicaciones tardías.Conclusiones: El uso de ópticas con canal de trabajo según técnicaSuPerLap posibilita prescindir de puertos adicionales en el tratamientode la hernia inguinal. Permite resultados funcionales comparables ycirugía sin cicatrices visibles. Minimiza la manipulación del cordónespermático. La laparoscopia permite el tratamiento concomitante dedefectos no diagnosticados previamente.

Introduction: Laparoscopic treatment of inguinal hernia is gainingpopularity in many hospitals, but the use of working channel scopes isnot as widely extended. We present our long-term experience with theSuPerLap (laparoscopic-assisted percutaneous suture) technique de-scribed by Rosell et al.(1) for epigastric hernia repair in the percutaneous,single-port treatment of inguinal hernia using working channel scopes.Materials and methods: A retrospective analysis of a series ofmale patients with congenital inguinal hernia undergoing surgery fromFebruary 2017 to December 2020 was carried out. A 5 mm-0º pleuro-scope with a 3.5 mm working channel, a 20 G epidural needle, a 36cm/3.5 mm laparoscopic Maryland dissector, and 3-0 polypropyleneand polyester sutures were used.Results: 384 inguinal hernia repairs using the SuPerLap techniquewere performed in 295 male patients – 206 unilateral repairs and 89bilateral repairs. In 24 bilateral cases (26.95%), preoperative diagnosishad been unilateral. Mean age was two years (2 weeks-13 years). Meanoperating time was 14 minutes (6-50 min) for unilateral repair, and27 minutes (14-80 min) for bilateral repair. There were two cases ofepigastric vessel damage, and one case of early recurrence in a new-born, who successfully underwent re-intervention using the SuPerLaptechnique. No late complications were recorded after a mean follow-upof 1-36 months.Conclusions: Working channel scopes using the SuPerLap tech-nique avoid additional ports in inguinal hernia repair. They allow forexcellent functional results, without visible scars, and minimize sper-matic cord manipulation. Laparoscopy allows previously undiagnoseddefects to be concomitantly treated.

Kid, a small protein of the parD stability system of plasmid R1, is an inhibitor of DNA replication acting at the initiation of DNA synthesis.

The Kid and Kis proteins are the killer component and the antagonist belonging to parD, a killer stability system of plasmid R1. The Kid and Kis proteins have been purified, the second one as a C-LYT-Kis fusion that conserves the antagonistic activity of the Kis protein, but not its auto-regulatory potential. Kid inhibits in vitro replication of CoEl to a basal level without altering the superhelicity of the template but it does not substantially affect in vitro replication of P4, a DnaA, DnaB, DnaC and DnaG-independent replicon. Kid inhibits lytic induction of a lambda, prophage, but this inhibition can be neutralized by excess DnaB. In addition, a multicopy dnaB recombinant, but not a multicopy dnaG recombinant, prevents the toxicity associated with this protein. Inhibition of ColE1 replication by Kid in vitro is prevented by the C-LYT-Kis protein. Functional analysis indicates that the antagonistic activity of Kis is independent of its activity as a co-regulator of the parD promoter. It is also shown that C-LYT-Kis and Kid interact, forming a tight complex. These results strongly suggest that the toxicity of the kid protein is due to inhibition of DnaB-dependent DNA replication, and that direct protein-protein interactions are involved in the neutralization of the activity of the killer protein by the antagonist.

Transcription of repA, the gene of the initiation protein of the Pseudomonas plasmid pPS10, is autoregulated by interactions of the RepA protein at a symmetrical operator.

Transcription of the repA gene of the Pseudomonas plasmid pPS10 is initiated from a sigma 70 type promoter located 81 bp upstream from the repA gene, extends through the repA gene and the adjacent open reading frame, and ends 1114 nucleotides downstream. The repA promoter is repressed by interactions of the RepA protein with a region of 44 bp that extends from the -10 box of the promoter to the dnaA box of the origin of replication. The core of the repA operator region is formed by two in-phase invertedly repeated sequences of 8 bp, S1 and S2, that flank the -35 box of the promoter, and that share homology with the internal sequences of the iterons present in the origin of replication. RepA enters at the operator region first by protein-DNA interactions and subsequently by protein-protein interactions. These sequential interactions lead to the formation of high, medium and low-mobility electrophoretic complexes. Formation of the high-order complexes seems to be important for an efficient repression of the promoter. Interactions of RepA with the repA promoter region (repPO) occur more efficiently than with the origin of replication.

Translating old drugs into new treatments: ribosomal frameshifting as a target for antiviral agents.

Programmed ribosomal frameshifting is used by many viruses to regulate the production of structural and enzymatic proteins. Altering the frameshifting efficiencies disrupts the virus life cycle and eliminates or reduces virus production. Ribosomal frameshifting therefore provides a unique target on which antiviral agents can function. This article describes a series of rapid assay strategies that have been developed and used to identify potential antiviral agents that target programmed -1 ribosomal frameshifting.

A mutation that decreases the efficiency of plasmid R1 replication leads to the activation of parD, a killer stability system of the plasmid.

The silent parD (kis/kid) stability operon of plasmid R1 is normally repressed by the co-ordinated action of the Kis and Kid proteins. In this report it is shown that a mutation in repA, the gene of the plasmid replication protein, that reduces two-fold the copy number of the plasmid, leads to the derepression of the parD system. This derepression can be prevented by a suppressor mutation in copB, a copy number control gene of plasmid R1, that increases the efficiency of replication of the repA mutant. Derepression of the wild-type parD system leads to high plasmid stability. These data show the activation of a plasmid stability operon by a mutation that reduces the efficiency of wild-type plasmid replication.

The RNA binding protein Pub1 modulates the stability of transcripts containing upstream open reading frames.

The nonsense-mediated mRNA decay (NMD) pathway functions to degrade transcripts containing nonsense codons. Transcripts containing mutations that insert an upstream open reading frame (uORF) in the 5'-UTR are degraded through NMD. However, several naturally occurring uORF-containing transcripts are resistant to NMD. Here we demonstrate that the GCN4 and YAP1 mRNAs, which contain uORFs, harbor a stabilizer element (STE) that prevents rapid NMD by interacting with the RNA binding protein Pub1. Conversely, a uORF-containing mRNA that lacks an STE, such as CPA1, is degraded by the NMD pathway. These results indicate that uORFs can play a pivotal role regulating both translation and turnover and that the Pub1p is a critical factor that modulates the stability of uORF-containing transcripts.

Utilizing the GCN4 leader region to investigate the role of the sequence determinants in nonsense-mediated mRNA decay.

In the yeast Saccharomyces cerevisiae, premature translation termination promotes rapid degradation of mRNAs. Accelerated decay requires the presence of specific cis-acting sequences which have been defined as downstream elements. It has been proposed that the role of the downstream element may be to promote translational reinitiation or ribosomal pausing. The GCN4 gene produces an mRNA that contains four short upstream open reading frames (uORFs) preceding the GCN4 protein-coding region in which translational initiation and reinitiation events occur. It was anticipated that these uORFs would function in a manner analogous to nonsense codons, promoting rapid degradation of the mRNA. However, the GCN4 transcript was not degraded by the nonsense-mediated mRNA decay pathway. We have investigated the role of the leader region of the GCN4 transcript in an effort to identify possible sequence elements that inactivate this decay pathway. We show that the GCN4 leader region does not harbor a downstream element needed to promote mRNA decay. In addition, using hybrid GCN4-PGK1 transcripts, we demonstrate that if a translational reinitiation signal precedes a downstream element, the mRNA will no longer be sensitive to nonsense-mediated decay. Furthermore, we demonstrate that the downstream element is functional only after a translational initiation and termination cycle has been completed but is unable to promote nonsense-mediated mRNA decay if it is situated 5' of a translational initiation site. Based on these results, the role of the downstream element will be discussed.

Structural and functional comparison between the stability systems ParD of plasmid R1 and Ccd of plasmid F.

The stability determined by the systems ParD of plasmid R1 and Ccd of plasmid F is due to the concerted action of two proteins, a cytotoxin and an antagonist of this function. In this paper we report that CcdA and Kis proteins, the antagonists of the Ccd and ParD systems respectively, share significant sequence homologies at both ends. In Kis, these regions seem to correspond to two different domains. Despite the structural similarities, Kis and CcdA are not interchangeable. In addition we have shown that the cytotoxins of these systems, the Kid and CcdB proteins, do not share structural homologies. In contrast to CcdB, the Kid protein of the ParD system induces RecA-dependent cleavage of the cI repressor of bacteriophage lambda very inefficiently or not at all. The functional implications of these results are discussed.

The kis and kid genes of the parD maintenance system of plasmid R1 form an operon that is autoregulated at the level of transcription by the co-ordinated action of the Kis and Kid proteins.

Stability mediated by the parD system of plasmid R1 is modulated by a killer protein, Kid, and by an antagonist of this function, Kis. Determination of the 5' ends of ParD transcripts, revealed that the genes coding for these proteins are transcribed from a single promoter. Analysis of the 3' end of the ParD RNAs indicated the existence of two transcripts: one of them coding for the Kis and Kid proteins, and the other coding only for Kis. Analysis of the effects of parD+ and kis+ recombinants on the beta-galactosidase levels expressed by different transcriptional and translational parD-lacZ fusions, and on the ParD RNA levels determined by a derepressed parD mutant, indicated that the Kis and Kid proteins repress coordinately the parD system at the transcriptional level. We discuss the relevance of these results in terms of the activities of the Kis and Kid proteins and in the context of the stabilization mediated by parD.

Translational coupling and limited degradation of a polycistronic messenger modulate differential gene expression in the parD stability system of plasmid R1.

The parD stability system of plasmid R1 is an auto-regulated operon containing two genes, kis and kid, that code, respectively, for a killer protein (Kid) and for an antagonist of Kid action (Kis protein). A polycistronic transcript and a shorter mRNA, coding only for Kis and ending in a stem-loop sequence, have been identified as the main parD transcripts in cells carrying a derepressed parD operon. In this communication we show that both parD mRNAs have a half-life close to 1 min and are present in similar amounts. Using an assay based on cell-free extracts of Escherichia coli, we demonstrate that the short kis mRNA originates from limited degradation of the bicistronic parD transcript and that the stem-loop structure within the 5' end of the kid gene is specifically required for the formation of this short transcript. In vivo experiments show that synthesis of Kis is required for efficient synthesis of Kid. These data indicate that RNA processing and translational coupling are important mechanisms that modulate the differential expression of the two genes, kis and kid, in the bicistronic parD operon.

Peptidyl-transferase inhibitors have antiviral properties by altering programmed -1 ribosomal frameshifting efficiencies: development of model systems.

The effects of two peptidyl-transferase inhibitors, anisomycin and sparsomycin, on ribosomal frameshifting efficiencies and the propagation of yeast double-stranded RNA viruses were examined. At sublethal doses in yeast cells these drugs specifically alter the efficiency of -1, but not of +1, ribosomal frameshifting. These compounds promote loss of the yeast L-A double-stranded RNA virus, which uses a programmed -1 ribosomal frameshift to produce its Gag-Pol fusion protein. Both of these drugs also change the efficiency of -1 ribosomal frameshifting in yeast and mammalian in vitro translation systems, suggesting that they may have applications to control the propagation of viruses of higher eukaryotes, which also use this translational regulatory mechanism. Our results offer a new set of antiviral agents that may potentially have a broad range of applications in the clinical, veterinary, and agricultural fields.

Should we kill the messenger? The role of the surveillance complex in translation termination and mRNA turnover.

Eukaryotes have evolved conserved mechanisms to rid cells of faulty gene products that can interfere with cell function. mRNA surveillance is an example of a pathway that monitors the translation termination process and promotes degradation of transcripts harboring premature translation termination codons. Studies on the mechanism of mRNA surveillance in yeast and humans suggest a common mechanism where a "surveillance complex" monitors the translation process and determines whether translation termination has occurred at the correct position within the mRNA. A model will be presented that suggests that the surveillance complex assesses translation termination by monitoring the transition of an RNP as it is converted from a nuclear to a cytoplasmic form during the initial rounds of translation.

Identifying the right stop: determining how the surveillance complex recognizes and degrades an aberrant mRNA.

The nonsense-mediated mRNA decay (NMD) pathway functions by checking whether translation termination has occurred prematurely and subsequently degrading the aberrant mRNAs. In Saccharomyces cerevisiae, it has been proposed that a surveillance complex scans 3' of the premature termination codon and searches for the downstream element (DSE), whose recognition by the complex identifies the transcript as aberrant and promotes its rapid decay. The results presented here suggest that translation termination is important for assembly of the surveillance complex. Neither the activity of the initiation ternary complex after premature translation termination has occurred nor the elongation phase of translation are essential for the activity of the NMD pathway. Once assembled, the surveillance complex is active for searching and recognizing a DSE for approximately 200 nt 3' of the stop codon. We have also identified a stabilizer sequence (STE) in the GCN4 leader region that inactivates the NMD pathway. Inactivation of the NMD pathway, as a consequence of either the DSE being too far from a stop codon or the presence of the STE, can be circumvented by inserting sequences containing a new translation initiation/termination cycle immediately 5' of the DSE. Further, the results indicate that the STE functions in the context of the GCN4 transcript to inactivate the NMD pathway.