Identification of mRNAs that undergo stop codon readthrough in Arabidopsis thaliana

A stop codon ensures termination of translation at a specific position on an mRNA. Sometimes, termination fails as translation machinery recognizes a stop codon as a sense codon. This leads to stop codon readthrough (SCR) resulting in the continuation of translation beyond the stop codon, generating protein isoforms with C-terminal extension. SCR has been observed in viruses, fungi, and multicellular organisms including mammals. However, SCR is largely unexplored in plants. In this study, we have analyzed ribosome profiling datasets to identify mRNAs that undergo SCR in Arabidopsis thaliana. Analyses of the ribosome density, ribosome coverage and three-nucleotide periodicity of the ribosome profiling reads, in the mRNA region downstream of the stop codon, provided strong evidence for SCR in mRNAs of 144 genes. This process generates putative peroxisomal targeting signal, nuclear localization signal, prenylation signal, transmembrane helix and intrinsically disordered regions in the C-terminal extension of several of these proteins. Gene ontology (GO) functional enrichment analysis revealed that these 144 genes belong to three major functional groups - translation, photosynthesis and abiotic stress tolerance. Finally, using a luminescence-based assay, we experimentally demonstrate SCR in representative mRNAs belonging to these functional classes. Based on these observations, we propose that SCR plays an important role in plant physiology by regulating the protein localization and function.

Selenium-Dependent Read Through of the Conserved 3’-Terminal UGA Stop Codon of HIV-1 nef

Objectives: The HIV-1 nef gene terminates in a 3’-UGA stop codon, which is highly conserved in the main group of HIV-1 subtypes, along with a downstream potential coding region that could extend the nef protein by 33 amino acids, if readthrough of the stop codon occurs. It has been proposed that antisense tethering interactions (ATIs) between a viral mRNA and a host selenoprotein mRNA are a potential viral strategy for the capture of a host selenocysteine insertion sequence (SECIS) element. This mRNA hijacking mechanism could enable the expression of virally encoded selenoprotein modules, through translation of in-frame UGA stop codons as selenocysteine (Sec). Here, our aim was to assess whether readthrough of the 3’-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells, and whether selenium-based mechanisms might be involved. Material and Methods: To assess UGA codon readthrough, we used fluorescence microscopy image analysis and flow cytometry of HEK 293 cells transfected with full length HIV-1 nef gene expression constructs including the 3’-UGA stop codon and a predicted thioredoxin reductase 1 (TXNRD1) antisense region spanning the UGA codon, engineered with a downstream in-frame green fluorescent protein (GFP) reporter gene. These were designed so that GFP can only be expressed by translational recoding of the UGA codon, that is, if the UGA codon is translated as an amino acid or bypassed by ribosomal hopping. To assess readthrough efficiency, appropriate mutant control constructs were used for 100% and 0% readthrough. We used anti-TXNRD1 siRNA to assess the possible role of the proposed antisense interaction in this event, by knockdown of TXNRD1 mRNA levels. Results: UGA stop codon readthrough efficiency for the wild-type nef construct was estimated by flow cytometry to be about 19% (P < 0.0001). siRNA knockdown of TXNRD1 mRNA resulted in a 67% decrease in GFP expression in this system relative to control cells (P < 0.0001), presumably due to reduced availability of the components involved in selenocysteine incorporation for the stop codon readthrough (i.e. the TXNRD1 SECIS element). Addition of 20 nM sodium selenite to the media enhanced stop codon readthrough in the pNefATI1 plasmid construct by >100% (P < 0.0001), that is, more than doubled the amount of readthrough product, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism. Conclusion: Our results show that readthrough of the 3’-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells, that this is dependent on selenium concentration, and the presence of TXNRD1 mRNA, supporting the proposed antisense tethering interaction.

Stop codon readthrough contexts influence reporter expression differentially depending on the presence of an IRES

Background: Previously we reported the discovery of stop codon readthrough in AMD1 mRNA followed by ribosome stalling at the end of a conserved Open Reading Frame (ORF) that we termed AMD1. To explain the severe suppression of reporters fused to AMD1 tail we proposed a mechanism invoking ribosome queueing. To test this hypothesis, we placed the reporter stop codon in the context of readthrough permissive sequences in a dual reporter vector with downstream reporter expression driven by the EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Methods: We employ dual luciferase assays, western blotting and RT-qPCR to explore the effects of test sequences downstream to the reporter stop codon on its expression in dual and monocistronic reporter vectors. Results: With the dual reporter system, the disproportionate reduction of upstream reporter activity is not specific to AMD1 tail and occurs as long as the readthrough stop codon context is present at the end of the reporter’s ORF. In a monocistronic vector without an IRES, the test sequences had distinct effects which were reflective of their properties e.g. AMD1 tail inhibitory effect. We further show with RT-qPCR that the EMCV IRES driven expression of a reporter is an accurate proxy of reporter RNA levels. Conclusions: While our findings provide little new information regarding the functional role of AMD1 tail, they raise caution for the use of viral IRES elements in expression vectors for studying mechanisms of mRNA translation. These findings may also be pertinent to the natural properties of read through permissive sequences and of IRES elements, though these require a separate investigation.

Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development

Background A number of cellular processes have evolved in metazoans that increase the proteome repertoire in relation to the genome, such as alternative splicing and translation recoding. Another such process, translational stop codon readthrough (SCR), generates C-terminally extended protein isoforms in many eukaryotes, including yeast, plants, insects, and humans. While comparative genome analyses have predicted the existence of programmed SCR in many species including humans, experimental proof of its functional consequences are scarce. Results We show that SCR of the Drosophila POU/Oct transcription factor Ventral veins lacking/Drifter (Vvl/Dfr) mRNA is prevalent in certain tissues in vivo, reaching a rate of 50% in the larval prothoracic gland. Phylogenetically, the C-terminal extension is conserved and harbors intrinsically disordered regions and amino acid stretches implied in transcriptional activation. Elimination of Vvl/Dfr translational readthrough by CRISPR/Cas9 mutagenesis changed the expression of a large number of downstream genes involved in processes such as chromatin regulation, neurogenesis, development, and immune response. As a proof-of-principle, we demonstrate that the C-terminal extension of Vvl/Dfr is necessary for correct timing of pupariation, by increasing the capacity to regulate its target genes. The extended Vvl/Dfr isoform acts in synergy with the transcription factor Molting defective (Mld) to increase the expression and biosynthesis of the steroid hormone ecdysone, thereby advancing pupariation. Consequently, late-stage larval development was prolonged and metamorphosis delayed in vvl/dfr readthrough mutants. Conclusions We demonstrate that translational recoding of a POU/Oct transcription factor takes place in a highly tissue-specific and temporally controlled manner. This dynamic and regulated recoding is necessary for normal expression of a large number of genes involved in many cellular and developmental processes. Loss of Vvl/Dfr translational readthrough negatively affects steroid hormone biosynthesis and delays larval development and progression into metamorphosis. Thus, this study demonstrates how SCR of a transcription factor can act as a developmental switch in a spatiotemporal manner, feeding into the timing of developmental transitions between different life-cycle stages. Graphical abstract

From Recoding to Peptides for MHC Class I Immune Display: Enriching Viral Expression, Virus Vulnerability and Virus Evasion

Many viruses, especially RNA viruses, utilize programmed ribosomal frameshifting and/or stop codon readthrough in their expression, and in the decoding of a few a UGA is dynamically redefined to specify selenocysteine. This recoding can effectively increase viral coding capacity and generate a set ratio of products with the same N-terminal domain(s) but different C-terminal domains. Recoding can also be regulatory or generate a product with the non-universal 21st directly encoded amino acid. Selection for translation speed in the expression of many viruses at the expense of fidelity creates host immune defensive opportunities. In contrast to host opportunism, certain viruses, including some persistent viruses, utilize recoding or adventitious frameshifting as part of their strategy to evade an immune response or specific drugs. Several instances of recoding in small intensively studied viruses escaped detection for many years and their identification resolved dilemmas. The fundamental importance of ribosome ratcheting is consistent with the initial strong view of invariant triplet decoding which however did not foresee the possibility of transitory anticodon:codon dissociation. Deep level dynamics and structural understanding of recoding is underway, and a high level structure relevant to the frameshifting required for expression of the SARS CoV-2 genome has just been determined.

Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.

Programmed Deviations of Ribosomes From Standard Decoding in Archaea

Genetic code decoding, initially considered to be universal and immutable, is now known to be flexible. In fact, in specific genes, ribosomes deviate from the standard translational rules in a programmed way, a phenomenon globally termed recoding. Translational recoding, which has been found in all domains of life, includes a group of events occurring during gene translation, namely stop codon readthrough, programmed ± 1 frameshifting, and ribosome bypassing. These events regulate protein expression at translational level and their mechanisms are well known and characterized in viruses, bacteria and eukaryotes. In this review we summarize the current state-of-the-art of recoding in the third domain of life. In Archaea, it was demonstrated and extensively studied that translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, and only one case of programmed –1 frameshifting has been reported so far in Saccharolobus solfataricus P2. However, further putative events of translational recoding have been hypothesized in other archaeal species, but not extensively studied and confirmed yet. Although this phenomenon could have some implication for the physiology and adaptation of life in extreme environments, this field is still underexplored and genes whose expression could be regulated by recoding are still poorly characterized. The study of these recoding episodes in Archaea is urgently needed.

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

Eukaryotic ribosome recognizes 3 context and stimulates stop codon readthrough

It is known that the nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3 contexts have been described that are unfavourable for translation termination; however, the exact molecular mechanism that mediates their effect remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3 stop codon contexts. Our results revealed that ribosomes can independently recognize certain contexts and ignore stop codons that are followed by these sequences. Moreover, the efficiency of translation termination at the weak 3 contexts was almost equal to the one at the standard context. We propose that weak 3 contexts interact with the 18S rRNA provoking a conformational change in the U-turn-like structure of the stop codon in the A site of ribosome. This change makes incorporation of the near-cognate tRNA more preferable than recognition of the stop codon by the release factors and increases readthrough.

A novel structural mechanism of ribosomal stop codon readthrough in VEGF-A

The process of ribosomal recoding is generally regulated by an autonomous mRNA signal downstream of stop-codons. While structural studies have provided mechanistic insights into viral systems, no such studies exist in mammalian systems. Here we define a novel structural mechanism for the VEGF-A readthrough system and show that regulation is multifaceted and complex, requiring a multipartite set of RNA elements located at long distances that interact with each other and with hnRNP A2/B1 to synergistically enhance readthrough levels. The Ax-element downstream of the stop codon adopts a unique multistem (SL-Ax1-3) architecture: SL-Ax1 interacts with hnRNP A2/B1, while SL-Ax2 interacts with an RNA element (SL-Au1) located ~500 nt upstream at the start of the coding sequence. SL-Au1 also independently binds to hnRNP A2/B1, which manipulates an equilibrium between alternate structures— from a sequestered bulge towards one that allows for the long-range interaction with SL-Ax2. Overall, our study not only highlights the significance of structural organization of elements within the coding sequence of mRNA, but also provides a functional relevance of the closed-loop mRNA organization in non-canonical translation and suggests complex mechanisms allow for finer integration of many signals for a required output.