RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression

The RECODE database is a compilation of 'programmed' translational recoding events taken from the scientific literature and personal communications. The database deals with programmed ribosomal frameshifting, codon redefinition and translational bypass occurring in a variety of organisms. The entries for each event include the sequences of the corresponding genes, their encoded proteins for both the normal and alternate decoding, the types of the recoding events involved, trans-factors and cis-elements that influence recoding. The database is freely available at http://recode.genetics. utah.edu/.     

Alternative fates of paused ribosomes during translation termination

The bacterial tmRNA·SmpB system facilitates recycling of stalled translational complexes in a process termed "ribosome rescue." During ribosome rescue, the nascent chain is tagged with the tmRNA-encoded ssrA peptide, which targets the tagged polypeptide for degradation. Translational pausing also induces a variety of recoding events such as frameshifts, ribosome hops, and stop codon readthrough. To examine the interplay between recoding and ribosome rescue, we determined the various fates of ribosomes that pause during translation termination. We expressed a model protein containing the C-terminal Asp-Pro nascent peptide motif (which interferes with translation termination) and quantified the protein chains produced by recoding and ssrA-peptide tagging. The nature and extent of translational recoding depended upon the codon for the C-terminal Pro residue, with CCU and CCC promoting efficient +1 frameshifting. In contrast, ssrA-peptide tagging was unaffected by C-terminal Pro coding. Moreover, +1 frameshifting was not suppressed by tmRNA·SmpB activity, suggesting that recoding and ribosome rescue are not competing events. However, cells lacking ribosomal protein L9 (ΔL9) exhibited a significant increase in recoding and a concomitant decrease in ssrA-peptide tagging. Pulse-chase analysis revealed that pre-termination ribosomes turn over more rapidly in ΔL9 cells, suggesting that increased recoding alleviates the translational arrest. Together, these results indicate that tmRNA·SmpB does not suppress transient ribosome pauses, but responds to prolonged translational arrest.     

Nonsense-mediated decay mutants do not affect programmed -1 frameshifting

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.     

Translational recoding in archaea

Translational recoding includes a group of events occurring during gene translation, namely stop codon readthrough, programmed ±1 frameshifting, and ribosome bypassing, which have been found in organisms from all domains of life. They serve to regulate protein expression at translational level and represent a relatively less known exception to the traditional central ‘dogma’ of biology that information flows as DNA→RNA→protein and that it is stored in a co-linear way between the 5′→3′ of nucleic acids and N→C-terminal of polypeptides. In archaea, in which translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, only one case of programmed ?1 frameshifting has been reported so far and further examples, although promising, have not been confirmed yet. We here summarize the current state-of-the-art of this field that, especially in archaea, has relevant implications for the physiology of life in extreme environments and for the origin of life.     

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.     

ATID: a web-oriented database for collection of publicly available alternative translational initiation events

SUMMARY: Alternative translational initiation is an important cellular mechanism contributing to the diversity of protein products and functions. We develop a database that provides a comprehensive collection of alternative translational initiation events. The purpose of this alternative translational initiation database (ATID) is to facilitate the systematic study of alternative translational initiation of genes. The current version of database contains 300 genes from Homo sapiens, Mus musculus and other species. Each of the genes has two or more isoforms due to alternative translational initiation. Resources in ATID, including gene information, alternative products of genes and domain structures of isoforms, are provided through a user-friendly web interface. AVAILABILITY: The ATID database is available for public use at http://bioinfo.au.tsinghua.edu.cn/atie/.     

A genome-wide survey of alternative translational initiation events in <Emphasis Type="Italic">Homo sapiens</Emphasis>

Alternative translational initiation is an important mechanism to increase the diversity of gene products. Although some of alternative translational initiation events have been reported, such information remains anecdotal and does not allow for any generalizations. The number of the known alternative translational initiation genes is so few that we know little about its mechanism. There is a great demand to discover more alternative translational initiation genes. However, it is arduously time-consuming to discover novel alternative translational initiation genes by the experimental method. Therefore we systematically analyzed protein sequences available in public database and predicted 1237 protein clusters as potential alternative translational initiation events. We concluded that about 8%–10% of human genes have alternative translational initiation sites. The results significantly increased the number of alternative translation initiation events and indicated that alternative translation initiation is an important and general regulation mechanism in the cellular process.     

Competition between SsrA tagging and translational termination at weak stop codons in Escherichia coli

SsrA is a tmRNA involved in tagging polypeptides on stalled ribosomes. The resulting fusion proteins are then degraded. We purified endogenous SsrA-tagged proteins by means of a genetically engineered SsrA and identified some of them. Analysis of the proteins suggested that they are tagged at their C-terminal extremities. One of them, ribokinase, is expressed from a messenger with a poorly efficient stop codon, leading to translational recoding events. A change in the ribokinase coding sequence from a weak to a strong translational stop sequence (UGAc to UAAu) annihilated SsrA tagging. Translational termination by UGA recruits the translational release factor (RF) 2. We observed that SsrA tagging of ribokinase was inversely correlated with RF2 activity, revealing a dynamic competition between translational termination and SsrA tagging.     

A genome-wide survey of alternative translational initiation events in Homo sapiens

Alternative translational initiation is an important mechanism to increase the diversity of gene products. Although some of alternative translational initiation events have been reported, such information remains anecdotal and does not allow for any generalizations. The number of the known alternative translational initiation genes is so few that we know little about its mechanism. There is a great demand to discover more alternative translational initiation genes. However, it is arduously time-consuming to discover novel alternative translational initiation genes by the experimental method. Therefore we systematically analyzed protein sequences available in public database and predicted 1237 protein clusters as potential alternative translational initiation events. We concluded that about 8%—10% of human genes have alternative translational initiation sites. The results significantly increased the number of alternative translation initiation events and indicated that alternative translation initiation is an important and general regulation mechanism in the cellular process.     

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.     

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.     

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.     

U2552 methylation at the ribosomal A-site is a negative modulator of translational accuracy

We have recently identified RrmJ, the first encoded protein of the rrmJ-ftsH heat shock operon, as being the Um(2552) methyltransferase of 23S rRNA, and reported that rrmJ-deficient strains exhibit growth defects, reduced translation rates and reduced stability of 70S ribosomes. U2552 is an ubiquitously methylated residue. It belongs to the A loop of 23S RNA which is an essential component of the ribosome peptidyltransferase centre and interacts directly with aminoacyl(A)-site tRNA. In the present study, we show that a lack of U2552 methylation, obtained in rrmJ-deficient mutants, results in a decrease in programmed +1 and -1 translational frameshifing and a decrease in readthrough of UAA and UGA stop codons. The increased translational accuracy of rrmJ-deficient strains suggests that the interaction between aminoacyl-tRNA and U2552 is important for selection of the correct tRNA at the ribosomal A site, and supports the idea that translational accuracy in vivo is optimal rather than maximal, thus pointing to the participation of recoding events in the normal cell physiology.     

Ribosomal protein L30 is a component of the UGA-selenocysteine recoding machinery in eukaryotes

The translational recoding of UGA as selenocysteine (Sec) is directed by a SECIS element in the 3' untranslated region (UTR) of eukaryotic selenoprotein mRNAs. The selenocysteine insertion sequence (SECIS) contains two essential tandem sheared G.A pairs that bind SECIS-binding protein 2 (SBP2), which recruits a selenocysteine-specific elongation factor and Sec-tRNA(Sec) to the ribosome. Here we show that ribosomal protein L30 is a component of the eukaryotic selenocysteine recoding machinery. L30 binds SECIS elements in vitro and in vivo, stimulates UGA recoding in transfected cells and competes with SBP2 for SECIS binding. Magnesium, known to induce a kink-turn in RNAs that contain two tandem G.A pairs, decreases the SBP2-SECIS complex in favor of the L30-SECIS interaction. We propose a model in which SBP2 and L30 carry out different functions in the UGA recoding mechanism, with the SECIS acting as a molecular switch upon protein binding.     

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.     

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.     

A second case of -1 ribosomal frameshifting affecting a major virion protein of the Lactobacillus bacteriophage A2

The bacteriophage A2 major tail protein gene utilizes a -1 translational frameshift to generate two structural polypeptides. Frameshifting is promoted by a slippery sequence and an RNA pseudoknot located 3' of the gene. The major head gene presents a similar recoding ability. A2 is the only phage described with two -1 frameshifts.     

Non-canonical decoding events at stop codons in eukaryotes

Regulation of protein synthesis at translation termination is a relatively under-explored, but rapidly expanding field. Recent advances in elucidating the mechanism of translation termination are helping to understand non-canonical events associated with translation termination. These "recoding" events include read-through of stop-codons, insertion of unusual amino acids such as selenocysteine and production of several polypeptides from one open reading frame. This review summarises data on termination-dependent recoding events, and proposes that there are two types of stop codon-associated sequences optimized to perform different functions: termination of translation per se or alternative elongation events.     

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.