Recoding Mechanism Research Articles

Exploring the Ribosome Helicase Activity in Context of Frameshifting using Single-Molecule Techniques

Although the structure of the bacterial ribosome, with varying resolutions has been uncovered, the subtle dynamics and interactions are to be pursued further. One vital aspect is the intrinsic helicase activity of the ribosome and how it unfolds structured mRNA during translation. The relevancy of the helicase function extends beyond general translation processes into recoding mechanisms such as frameshifting. Such process is found in viruses, bacteria and eukaryotes at varying efficiencies related to both physiological and pathological functions posing an interest to explore it further. Thus, we aim to study this process using single-molecule approaches along with bulk methods. First, we are developing bifluorescent constructs to quantify frameshifting both in vitro and in vivo for bacteria. Also, we are developing an ultrahigh resolution optical tweezers with fluorescence detection capabilities. The tweezers - FRET setup along with different labeling schemes could be used to monitor the ribosome during translation and its interactions with mRNA. This could elucidate further the dynamics and conformational changes intrinsically within the ribosome during the process of unfolding. In addition, it could provide fundamental insight into the variable efficiency of different frameshifting signals whether through the thermodynamic stability or conformational changes of structured elements within the signal.

Recoding the Genetic Code with Selenocysteine

Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as the Sec insertion machinery was found to be able to site-specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid (AA) to Sec was observed along with a tenfold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high-fidelity sense-codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl-tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell.

A dynamical model of programmed −1 ribosomal frameshifting

Programmed −1 ribosomal frameshifting is the most widely used translational recoding mechanism of RNA viruses. How the frameshifting occurs at the slippery sequence on the presence of a downstream mRNA pseudoknot has not been fully understood. Here, we present systematical analysis of the –1 frameshifting that can occur during every transition step in elongation phase of protein synthesis by Escherichia coli ribosomes, showing that the –1 frameshifting can occur mainly during three periods. One is during translocation step, another period is after the posttranslocation and before the binding of the ternary complex aminoacyl-tRNA.EF-Tu.GTP, and the third period is after codon recognition and before peptidyl transfer. Of the three periods, the translocation step makes the most contribution to the –1 frameshifting. During the translocation step when the mRNA channel is open, due to the presence of energy barrier to the forward translocation of the ribosome along the mRNA template, which results from unwinding of the mRNA pseudoknot, the reverse ribosomal rotation from ratcheted to non-ratcheted conformation induces the frameshifting and futile translocation besides the effective forward translocation. During the other two slow periods when the mRNA channel is tight, the annealing of the unwound base pair in the mRNA pseudoknot can also promote the –1 frameshifting. Our theoretical results provide a consistent explanation of a lot of independent experimental data and also provide predicted results.

A Genome-Wide Analysis of RNA Pseudoknots That Stimulate Efficient −1 Ribosomal Frameshifting or Readthrough in Animal Viruses

Programmed −1 ribosomal frameshifting (PRF) and stop codon readthrough are two translational recoding mechanisms utilized by some RNA viruses to express their structural and enzymatic proteins at a defined ratio. Efficient recoding usually requires an RNA pseudoknot located several nucleotides downstream from the recoding site. To assess the strategic importance of the recoding pseudoknots, we have carried out a large scale genome-wide analysis in which we used an in-house developed program to detect all possible H-type pseudoknots within the genomic mRNAs of 81 animal viruses. Pseudoknots are detected downstream from ~85% of the recoding sites, including many previously unknown pseudoknots. ~78% of the recoding pseudoknots are the most stable pseudoknot within the viral genomes. However, they are not as strong as some designed pseudoknots that exhibit roadblocking effect on the translating ribosome. Strong roadblocking pseudoknots are not detected within the viral genomes. These results indicate that the decoding pseudoknots have evolved to possess optimal stability for efficient recoding. We also found that the sequence at the gag-pol frameshift junction of HIV1 harbors potential elaborated pseudoknots encompassing the frameshift site. A novel mechanism is proposed for possible involvement of the elaborated pseudoknots in the HIV1 PRF event.

C-Jun Amino-Terminal Kinase-1 Mediates Glucose-Responsive Upregulation of the RNA Editing Enzyme ADAR2 in Pancreatic Beta-Cells

A-to-I RNA editing catalyzed by the two main members of the adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, represents a RNA-based recoding mechanism implicated in a variety of cellular processes. Previously we have demonstrated that the expression of ADAR2 in pancreatic islet β-cells is responsive to the metabolic cues and ADAR2 deficiency affects regulated cellular exocytosis. To investigate the molecular mechanism by which ADAR2 is metabolically regulated, we found that in cultured β-cells and primary islets, the stress-activated protein kinase JNK1 mediates the upregulation of ADAR2 in response to changes of the nutritional state. In parallel with glucose induction of ADAR2 expression, JNK phosphorylation was concurrently increased in insulin-secreting INS-1 β-cells. Pharmacological inhibition of JNKs or siRNA knockdown of the expression of JNK1 prominently suppressed glucose-augmented ADAR2 expression, resulting in decreased efficiency of ADAR2 auto-editing. Consistently, the mRNA expression of Adar2 was selectively reduced in the islets from JNK1 null mice in comparison with that of wild-type littermates or JNK2 null mice, and ablation of JNK1 diminished high-fat diet-induced Adar2 expression in the islets from JNK1 null mice. Furthermore, promoter analysis of the mouse Adar2 gene identified a glucose-responsive region and revealed the transcription factor c-Jun as a driver of Adar2 transcription. Taken together, these results demonstrate that JNK1 serves as a crucial component in mediating glucose-responsive upregulation of ADAR2 expression in pancreatic β-cells. Thus, the JNK1 pathway may be functionally linked to the nutrient-sensing actions of ADAR2-mediated RNA editing in professional secretory cells.

The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element

Selenocysteine insertion into selenoproteins involves the translational recoding of UGA stop codons. In mammals, selenoprotein expression further depends on selenium availability, which has been particularly described for glutathione peroxidase 1 and 4 (Gpx1 and Gpx4). The SECIS element located in the 3′UTR of the selenoprotein mRNAs is a modulator of UGA recoding efficiency in adequate selenium conditions. One of the current models for the UGA recoding mechanism proposes that the SECIS binds SECIS-binding protein 2 (SBP2), which then recruits a selenocysteine-specific elongation factor (EFsec) and tRNASec to the ribosome, where L30 acts as an anchor. The involvement of the SECIS in modulation of UGA recoding activity was investigated, together with SBP2 and EFsec, in Hek293 cells cultured with various selenium levels. Luciferase reporter constructs, in transiently or stably expressing cell lines, were used to analyze the differential expression of Gpx1 and Gpx4. We showed that, upon selenium fluctuation, the modulation of UGA recoding efficiency depends on the nature of the SECIS, with Gpx1 being more sensitive than Gpx4. Attenuation of SBP2 and EFsec levels by shRNAs confirmed that both factors are essential for efficient selenocysteine insertion. Strikingly, in a context of either EFsec or SBP2 attenuation, the decrease in UGA recoding efficiency is dependent on the nature of the SECIS, GPx1 being more sensitive. Finally, the profusion of selenium of the culture medium exacerbates the lack of factors involved in selenocysteine insertion.

Stimulation of ribosomal frameshifting by antisense LNA

Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12–18 nt. Antisense oligonucleotides bearing locked nucleid acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.

Single-Molecule FRET Studies on Frameshifting RNA Structures of Human Immunodeficiency Virus

Correspondence between DNA sequences and its protein product is not straightforward because the genetic information can be dynamically reprogrammed during various steps of gene-tic decoding process. The programmed -1 ribosomal frameshift-ing (-1PRF) is an example of such genetic recoding mechanisms, where the reading frame of the translation machinery is shifted toward -1 direction at a predetermined position of messenger RNA.Biochemical studies for more than last 20 years have eluci-dated that -1PRF occurs during the interaction of the ribosome with RNA structures, positioned 6 ~ 8 bases downstream from the frameshifting site. Except rare cases, RNA pseudoknots are the RNA structure to induce -1PRF.

Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression

Selenium, an essential trace element, is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. In order to synthesize selenoproteins, a translational reprogramming event must occur since Sec is encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3′ untranslated region of the transcript. The SECIS acts as a platform for RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV crosslinking, we identified a 110 kDa protein, which binds with high affinity to SECIS elements from a subset of selenoprotein mRNAs. The crosslinking activity was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis. In vitro binding assays showed that purified nucleolin discriminates among SECIS elements in the absence of other factors. Based on siRNA experiments, nucleolin is required for the optimal expression of certain selenoproteins. There was a good correlation between the affinity of nucleolin for a SECIS and its effect on selenoprotein expression. As selenoprotein transcript levels and localization did not change in siRNA-treated cells, our results suggest that nucleolin selectively enhances the expression of a subset of selenoproteins at the translational level.

A homogeneous cell-based bicistronic fluorescence assay for high-throughput identification of drugs that perturb viral gene recoding and read-through of nonsense stop codons

Recoding mechanisms are programmed protein synthesis events used commonly by viruses but only very rarely in cells for cellular gene expression. For example, HIV-1 has an absolute reliance on frameshifting to produce the correct ratio of key proteins critical for infectivity. To exploit such recoding sites as therapeutic targets, a simple homogeneous assay capable of detecting small perturbations in these low-frequency (<5%) events is required. Current assays based on dual luciferase reporters use expensive substrates and are labor-intensive, both impediments for high-throughput screening. We have developed a cell-based bifluorophore assay able to measure accurately small recoding changes (<0.1%) with a high Z'-factor in 24- or 96-well formats that could be extended to 384 wells. In cases of nonsense mutations arising within coding regions of genes, the assay is suitable for assessing the potential of screened compounds to increase read-through at these nonprogrammed stop signals of variable termination efficiency.

Stop codon recoding mechanism revealed by the suppressor tRNAPyl/PylS complex structure

Stop codon recoding mechanism revealed by the suppressor tRNAPyl/PylS complex structure

A-to-I RNA editing alters less-conserved residues of highly conserved coding regions: Implications for dual functions in evolution

The molecular mechanism and physiological function of recoding by A-to-I RNA editing is well known, but its evolutionary significance remains a mystery. We analyzed the RNA editing of the Kv2 K(+) channel from different insects spanning more than 300 million years of evolution: Drosophila melanogaster, Culex pipiens (Diptera), Pulex irritans (Siphonaptera), Bombyx mori (Lepidoptera), Tribolium castaneum (Coleoptera), Apis mellifera (Hymenoptera), Pediculus humanus (Phthiraptera), and Myzus persicae (Homoptera). RNA editing was detected across all Kv2 orthologs, representing the most highly conserved RNA editing event yet reported in invertebrates. Surprisingly, five of these editing sites were conserved in squid (Mollusca) and were possibly of independent origin, suggesting phylogenetic conservation of editing between mollusks and insects. Based on this result, we predicted and experimentally verified two novel A-to-I editing sites in squid synaptotagmin I transcript. In addition, comparative analysis indicated that RNA editing usually occurred within highly conserved coding regions, but mostly altered less-conserved coding positions of these regions. Moreover, more than half of these edited amino acids are genomically encoded in the orthologs of other species; an example of a conversion model of the nonconservative edited site is addressed. Therefore, these data imply that RNA editing might play dual roles in evolution by extending protein diversity and maintaining phylogenetic conservation.

Identification of Hepta- and Octo-Uridine stretches as sole signals for programmed +1 and -1 ribosomal frameshifting during translation of SARS-CoV ORF 3a variants

Programmed frameshifting is one of the translational recoding mechanisms that read the genetic code in alternative ways. This process is generally programmed by signals at defined locations in a specific mRNA. In this study, we report the identification of hepta- and octo-uridine stretches as sole signals for programmed +1 and −1 ribosomal frameshifting during translation of severe acute respiratory syndrome coronavirus (SARS-CoV) ORF 3a variants. SARS-CoV ORF 3a encodes a minor structural protein of 274 amino acids. Over the course of cloning and expression of the gene, a mixed population of clones with six, seven, eight and nine T stretches located 14 nt downstream of the initiation codon was found. In vitro and in vivo expression of clones with six, seven and eight Ts, respectively, showed the detection of the full-length 3a protein. Mutagenesis studies led to the identification of the hepta- and octo-uridine stretches as slippery sequences for efficient frameshifting. Interestingly, no stimulatory elements were found in the sequences upstream or downstream of the slippage site. When the hepta- and octo-uridine stretches were used to replace the original slippery sequence of the SARS-CoV ORF 1a and 1b, efficient frameshift events were observed. Furthermore, the efficiencies of frameshifting mediated by the hepta- and octo-uridine stretches were not affected by mutations introduced into a downstream stem–loop structure that totally abolish the frameshift event mediated by the original slippery sequence of ORF 1a and 1b. Taken together, this study identifies the hepta- and octo-uridine stretches that function as sole elements for efficient +1 and −1 ribosomal frameshift events.

Intronic breakpoint definition and transcription analysis in DMD/BMD patients with deletion/duplication at the 5′ mutation hot spot of the dystrophin gene

Dystrophin mutations occurring at the 5′ end of the gene frequently behave as exceptions to the “frame rule,” their clinical severity being variable and often not related to the perturbation of the translation reading frame. The molecular mechanisms underlying the phenotypic variability of 5′ dystrophin mutations have not been fully clarified. We have characterized the genomic breakpoints within introns 2, 6 and 7 and identified the splicing profiles in a cohort of DMD/BMD patients with deletion of dystrophin exons 3–7, 3–6 and duplication of exons 2–4.Our findings indicate that the occurrence of intronic cryptic promoter as well as corrective splicing events are unlikely to play a role in exons 3–7 deleted patients phenotypic variability. Our data suggest that re-initiation of translation could represent a major mechanism responsible for the production of a residual dystrophin in some patients with exons 3–7 deletion.Furthermore, we observed that the out-of-frame exon 2a is almost constantly spliced into a proportion of the dystrophin transcripts in the analysed patients. In the exons 2–4 duplicated DMD patient, producing both in-frame and out-of-frame transcripts, this splicing behaviour might represent a critical factor contributing to the severe phenotype. In conclusion, we suggest that multiple mechanisms may have a role in modulating the outcome of 5′ dystrophin mutations, including recoding mechanisms and unusual splicing choices.

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.

Translational recoding signals between gag and pol in diverse LTR retrotransposons.

Because of their compact genomes, retroelements (including retrotransposons and retroviruses) employ a variety of translational recoding mechanisms to express Gag and Pol. To assess the diversity of recoding strategies, we surveyed gag/pol gene organization among retroelements from diverse host species, including elements exhaustively recovered from the genome sequences of Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Candida albicans, and Arabidopsis thaliana. In contrast to the retroviruses, which typically encode pol in the -1 frame relative to gag, nearly half of the retroelements surveyed encode a single gag-pol open reading frame. This was particularly true for the Ty1/copia group retroelements. Most animal Ty3/gypsy retroelements, on the other hand, encode gag and pol in separate reading frames, and likely express Pol through +1 or -1 frameshifting. Conserved sequences conforming to slippery sites that specify viral ribosomal frameshifting were identified among retroelements with pol in the -1 frame. None of the plant retroelements encoded pol in the -1 frame relative to gag; however, two closely related plant Ty3/gypsy elements encode pol in the +1 frame. Interestingly, a group of plant Ty1/copia retroelements encode pol either in a +1 frame relative to gag or in two nonoverlapping reading frames. These retroelements have a conserved stem-loop at the end of gag, and likely express pol either by a novel means of internal ribosomal entry or by a bypass mechanism.

Nonword reading across orthographies: How flexible is the choice of reading units?

It was predicted that children learning to read inconsistent orthographies (e.g., English) should show considerable flexibility in making use of spelling–sound correspondences at different unit sizes whereas children learning to read consistent orthographies (e.g., German) should mainly employ small-size grapheme–phoneme strategies. This hypothesis was tested in a cross-language blocking experiment using nonwords that could only be read using small-size grapheme–phoneme correspondences (small-unit nonwords) and phonologically identical nonwords that could be decoded using larger correspondences (large-unit nonwords). These small-unit and large-unit nonwords were either presented mixed together in the same lists or blocked by unit size. It was found that English children, but not German children, showed blocking effects (better performance when items were blocked by nonword type than in mixed lists). This suggests that in mixed lists, English readers have to switch back and forth between small-unit and large-unit processing, resulting in switching costs. These results are interpreted in terms of differences concerning the grain size of the phonological recoding mechanisms developed by English and German children.

Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting

RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49°, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.

Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display.

An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.

Translational Recoding Induced by G-Rich mRNA Sequences That Form Unusual Structures

We investigated a herpesvirus mutant that contains a single base insertion in its thymidine kinase ( tk) gene yet expresses low levels of TK via a net +1 translational recoding event. Within this mutant gene, we defined a G-rich signal that is sufficient to induce recoding. Unlike other translational recoding events, downstream RNA structures or termination codons did not stimulate recoding, and paused ribosomes were not detected. Mutational analysis indicated that specific tRNAs or codon–anticodon slippage were unlikely to account for recoding. Rather, recoding efficiency correlated with the G-richness of the signal and its ability to form unusual structures. These findings identify a mechanism of translational recoding with unique features and potential implications for clinical drug resistance and other biological systems.