Large ribosomal protein 4 increases efficiency of viral recoding sequences

Expression of retroviral replication enzymes (Pol) requires a controlled translational recoding event to bypass the stop codon at the end of gag. This recoding event occurs either by direct suppression of termination via the insertion of an amino acid at the stop codon (readthrough) or by alteration of the mRNA reading frame (frameshift). Here we report the effects of a host protein, large ribosomal protein 4 (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we found that transfection of cells with a plasmid encoding RPL4 cDNA increases recoding efficiency in a dose-dependent manner, with a maximal enhancement of nearly twofold. Expression of RPL4 increases recoding of reporters containing retroviral readthrough and frameshift sequences, as well as the Sindbis virus leaky termination signal. RPL4-induced enhancement of recoding is cell line specific and appears to be specific to RPL4 among ribosomal proteins. Cotransfection of RPL4 cDNA with Moloney murine leukemia proviral DNA results in Gag processing defects and a reduction of viral particle formation, presumably caused by the RPL4-dependent alteration of the Gag-to-Gag-Pol ratio required for virion assembly and release.     

The gene of an archaeal alpha-L-fucosidase is expressed by translational frameshifting

The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a alpha-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a -1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed -1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea.     

Recoding in archaea

Standard decoding of the genetic information into polypeptides is performed by one of the most sophisticated cell machineries, the translating ribosome, which, by following the genetic code, ensures the correspondence between the mature mRNA and the protein sequence. However, the expression of a minority of genes requires programmed deviations from the standard decoding rules, globally named recoding. This includes ribosome programmed -/+1 frameshifting, ribosome hopping, and stop codon readthrough. Recoding in Archaea was unequivocally demonstrated only for the translation of the UGA stop codon into the amino acid selenocysteine. However, a new recoding event leading to the 22nd amino acid pyrrolysine and the preliminary reports on a gene regulated by programmed -1 frameshifting have been recently described in Archaea. Therefore, it appears that the study of this phenomenon in Archaea is still at its dawn and that most of the genes whose expression is regulated by recoding are still uncharacterized.     

UGA: a dual signal for 'stop' and for recoding in protein synthesis

UGA remains an enigma as a signal in protein synthesis. Long recognized as a stop signal that is prone to failure when under competition from near cognate events, there was growing belief that there might be functional significance in the production of small amounts of extended proteins. This view has been reinforced with the discovery that UGA is found at some recoding sites where frameshifting occurs as a regulatory mechanism for controlling the gene expression of specific proteins, and it also serves as the code for selenocysteine (Sec), the 21st amino acid. Why does UGA among the stop signals play this role specifically, and how does it escape being used to stop protein synthesis efficiently at recoding sites involving Sec incorporation or shifts to a new translational frame? These issues concerning the UGA stop signals are discussed in this review.     

Versatile vectors to study recoding: conservation of rules between yeast and mammalian cells.

In many viruses and transposons, expression of some genes requires alternative reading of the genetic code, also called recoding. Such events depend on specific mRNA sequences and can lead to read through of an in-frame stop codon or to +1 or -1 frameshifting. Here, we addressed the issue of conservation of recoding rules between the yeast Saccharomyces cerevisiae and mammalian cells by establishing a versatile vector that can be used to study recoding in both species. We first assessed this vector by analysing the site of +1 frameshift of the Ty1 transposon. Two sequences from higher organisms were then tested in both yeast and mammalian cells: the gag-pol junction of human immunodeficiency virus type 1 (HIV-1) (a site of -1 frameshift), and the stop codon region of the replicase cistron from the tobacco mosaic virus (a site of UAG read through). We show that both sequences direct a high level of recoding in yeast. Furthermore, different mutations of the target sequences have similar effects on recoding in yeast and in mouse cells. Most notably, a strong decrease of frameshifting was observed in the absence of the HIV-1 stem-loop stimulatory signal. Taken together, these data suggest that mechanisms of some recoding events are conserved between lower and higher eukaryotes, thus allowing the use of S. cerevisiae as a model system to study recoding on target sequences from higher organisms.     

Identification and characterisation of a developmentally regulated mammalian gene that utilises -1 programmed ribosomal frameshifting

Translational recoding of mRNA through a –1 ribosomal slippage mechanism has been observed in RNA viruses and retrotransposons of both eukaryotes and prokaryotes. Whilst this provides a potentially powerful mechanism of gene regulation, the utilization of –1 translational frameshifting in regulating mammalian gene expression has remained obscure. Here we report a mammalian gene, Edr, which provides the first example of –1 translational recoding in a eukaryotic cellular gene. In addition to bearing functional frameshift elements that mediate expression of distinct polypeptides, Edr bears both CCHC zinc-finger and putative aspartyl protease catalytic site retroviral-like motifs, indicative of a relic retroviral-like origin for Edr. These features, coupled with conservation of Edr as a single copy gene in mouse and man and striking spatio-temporal regulation of expression during embryogenesis, suggest that Edr plays a functionally important role in mammalian development.     

Predominance of six different hexanucleotide recoding signals 3' of read-through stop codons

Redefinition of UAG, UAA and UGA to specify a standard amino acid occurs in response to recoding signals present in a minority of mRNAs. This ‘read-through’ is in competition with termination and is utilized for gene expression. One of the recoding signals known to stimulate read-through is a hexanucleotide sequence of the form CARYYA 3′ adjacent to the stop codon. The present work finds that of the 91 unique viral sequences annotated as read-through, 90% had one of six of the 64 possible codons immediately 3′ of the read-through stop codon. The relative efficiency of these read-through contexts in mammalian tissue culture cells has been determined using a dual luciferase fusion reporter. The relative importance of the identity of several individual nucleotides in the different hexanucleotides is complex.     

A dynamic competition between release factor 2 and the tRNA(Sec) decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H.

Factors affecting competition between termination and elongation in vivo during translation of the fdhF selenocysteine recoding site (UGA) were studied with wild-type and modified fdhF sequences. Altering sequences surrounding the recoding site UGA without affecting RNA secondary structure indicated that the kinetics of stop signal decoding have a significant influence on selenocysteine incorporation efficiency. The UGA in the wild-type fdhF sequence remains 'visible' to the factor and forms a site-directed cross-link when mRNA stem-loop secondary structure is absent, but not when it is present. The timing of the secondary structure unfolding during translation may be a critical feature of competition between release factor 2 and tRNA(Sec) for decoding UGA. Increasing the cellular concentration of either of these decoding molecules for termination or selenocysteine incorporation showed that they were able to compete for UGA by a kinetic competition that is dynamic and dependent on the Escherichia coli growth rate. The tRNA(Sec)-mediated decoding can compete more effectively for the UGA recoding site at lower growth rates, consistent with anaerobic induction of fdhF expression.     

Selective Up-regulation of Human Selenoproteins in Response to Oxidative Stress

Selenocysteine is inserted into selenoproteins via the translational recoding of a UGA codon, normally used as a stop signal. This process depends on the nature of the selenocysteine insertion sequence element located in the 3′ UTR of selenoprotein mRNAs, selenium bioavailability, and, possibly, exogenous stimuli. To further understand the function and regulation of selenoproteins in antioxidant defense and redox homeostasis, we investigated how oxidative stress influences selenoprotein expression as a function of different selenium concentrations. We found that selenium supplementation of the culture media, which resulted in a hierarchical up-regulation of selenoproteins, protected HEK293 cells from reactive oxygen species formation. Furthermore, in response to oxidative stress, we identified a selective up-regulation of several selenoproteins involved in antioxidant defense (Gpx1, Gpx4, TR1, SelS, SelK, and Sps2). Interestingly, the response was more efficient when selenium was limiting. Although a modest change in mRNA levels was noted, we identified a novel translational control mechanism stimulated by oxidative stress that is characterized by up-regulation of UGA-selenocysteine recoding efficiency and relocalization of SBP2, selenocysteine-specific elongation factor, and L30 recoding factors from the cytoplasm to the nucleus.     

Pseudogene recoding revealed from proteomic analysis of salmonella serovars

Recoding refers to the reprogramming of mRNA translation by nonstandard read-out rules. In this study, we used stable isotope labeling with amino acids in cell culture (SILAC) technology to investigate the proteome of host-adapted Salmonella serovars, which are characteristic of accumulation of pseudogenes. Interestingly, a few annotated pseudogenes were indeed able to express peptides downstream of the inactivation site, suggesting the occurrence of recoding. Two mechanisms of recoding, namely, programmed frameshifting and codon redefinition, were both found. We believe that the phenomena of recoding are not rare in bacteria. More studies are required for a better understanding of bacterial translation and the implication of pseudogene recoding in Salmonella serovars.     

Polyamine sensing during antizyme mRNA programmed frameshifting

A key regulator of cellular polyamine levels from yeasts to mammals is the protein antizyme. The antizyme gene consists of two overlapping reading frames with ORF2 in the +1 frame relative to ORF1. A programmed +1 ribosomal frameshift occurs at the last codon of ORF1 and results in the production of full-length antizyme protein. The efficiency of frameshifting is proportional to the concentration of polyamines, thus creating an autoregulatory circuit for controlling polyamine levels. The mRNA recoding signals for frameshifting include an element 5' and a pseudoknot 3' of the shift site. The present work illustrates that the ORF1 stop codon and the 5' element are critical for polyamine sensing, whereas the 3' pseudoknot acts to stimulate frameshifting in a polyamine independent manner. We also demonstrate that polyamines are required to stimulate stop codon readthrough at the MuLV redefinition site required for normal expression of the GagPol precursor protein.     

Translational Redefinition of UGA Codons Is Regulated by Selenium Availability

Incorporation of selenium into ∼25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA^[Ser]Sec Um34 methylation. Furthermore, we find evidence for translation in the 5′-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins.     

Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine

The selenocysteine insertion sequence (SECIS) element directs the translational recoding of UGA as selenocysteine. In eukaryotes, the SECIS is located downstream of the UGA codon in the 3′-UTR of the selenoprotein mRNA. Despite poor sequence conservation, all SECIS elements form a similar stem-loop structure containing a putative kink-turn motif. We functionally characterized the 26 SECIS elements encoded in the human genome. Surprisingly, the SECIS elements displayed a wide range of UGA recoding activities, spanning several 1000-fold in vivo and several 100-fold in vitro. The difference in activity between a representative strong and weak SECIS element was not explained by differential binding affinity of SECIS binding Protein 2, a limiting factor for selenocysteine incorporation. Using chimeric SECIS molecules, we identified the internal loop and helix 2, which flank the kink-turn motif, as critical determinants of UGA recoding activity. The simultaneous presence of a GC base pair in helix 2 and a U in the 5′-side of the internal loop was a statistically significant predictor of weak recoding activity. Thus, the SECIS contains intrinsic information that modulates selenocysteine incorporation efficiency.     

Altered expression of plant lysyl tRNA synthetase promotes tRNA misacylation and translational recoding of lysine

The Arabidopsis thaliana lysyl tRNA synthetase (AtKRS) structurally and functionally resembles the well-characterized prokaryotic class IIb KRS, including the propensity to aminoacylate tRNA(Lys) with suboptimal identity elements, as well as non-cognate tRNAs. Transient expression of AtKRS in carrot cells promotes aminoacylation of such tRNAs in vivo and translational recoding of lysine at nonsense codons. Stable expression of AtKRS in Zea mays causes translational recoding of lysine into zeins, significantly enriching the lysine content of grain.     

RECODE 2003

The RECODE database is a compilation of translational recoding events (programmed ribosomal frameshifting, codon redefinition and translational bypass). The database provides information about the genes utilizing these events for their expression, recoding sites, stimulatory sequences and other relevant information. The Database is freely available at http://recode.genetics.utah.edu/.     

Maintaining the ribosomal reading frame: the influence of the E site during translational regulation of release factor 2

Maintenance of the translation reading frame is one of the most remarkable achievements of the ribosome while decoding the information of an mRNA. Loss of the reading frame through spontaneous frameshifting occurs with a frequency of one in 30,000 amino acid incorporations. However, at many recoding sites, the mechanism that controls reading frame maintenance is switched off. One such example is the programmed +1 frameshift site of the prfB gene encoding the termination factor RF2, in which slippage into the forward frame by one nucleotide can attain an efficiency of approximately 100%, namely, four orders of magnitude higher than normally observed. Here, using the RF2 frameshift window, we demonstrate that premature release of the E site tRNA from the ribosome is coupled with high-level frameshifting. Consistently, in a minimal system, the presence of the E site tRNA prevents the +1 frameshift event, illustrating the importance of the E site for reading-frame maintenance.     

Structure of prokaryotic SECIS mRNA hairpin and its interaction with elongation factor SelB

In prokaryotes, the recoding of a UGA stop codon as a selenocysteine codon requires a special elongation factor (EF) SelB and a stem-loop structure within the mRNA called a selenocysteine insertion sequence (SECIS). Here, we used NMR spectroscopy to determine the solution structure of the SECIS mRNA hairpin and characterized its interaction with the mRNA-binding domain of SelB. Our structural and biochemical data identified the conserved structural features important for binding to EF SelB within different SECIS RNA sequences. In the free SECIS mRNA structure, conserved nucleotides are strongly exposed for recognition by SelB. Binding of the C-terminal domain of SelB stabilizes the RNA secondary structure. In the protein-RNA complex, a Watson-Crick loop base-pair leaves a GpU sequence accessible for SelB recognition. This GpU sequence at the tip of the capping tetraloop and a bulge uracil five Watson-Crick base-pairs apart from the GpU are essential for interaction with SelB.     

Expression of macrophage asialoglycoprotein-binding protein is induced through MAPK classical pathway

Macrophage asialoglycoprotein-binding protein (M-ASGP-BP) is a Gal/GalNAc-specific lectin, which functions as an endocytosis receptor. We found that the expression of M-ASGP-BP mRNA in bone marrow cells was induced during the differentiation into macrophages. To investigate the mechanism by which M-ASGP-BP mRNA expression is induced, we used U937 cells as a model. Treatment of U937 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in M-ASGP-BP mRNA expression within 6 h. This induction was completely inhibited by PKC inhibitors, calphostin C, and staurosporine. Furthermore, MAP kinase inhibitors PD98059, but not SB202190, blocked M-ASGP-BP mRNA expression. These data indicate that M-ASGP-BP mRNA expression occurs through the activation of PKC and the MAPK classical pathway in the course of cell differentiation into macrophages.