Evolution of Virus-like Features and Intrinsically Disordered Regions in Retrotransposon-derived Mammalian Genes.

Several mammalian genes have originated from the domestication of retrotransposons, selfish mobile elements related to retroviruses. Some of the proteins encoded by these genes have maintained virus-like features; including self-processing, capsid structure formation, and the generation of different isoforms through -1 programmed ribosomal frameshifting. Using quantitative approaches in molecular evolution and biophysical analyses, we studied 28 retrotransposon-derived genes, with a focus on the evolution of virus-like features. By analyzing the rate of synonymous substitutions, we show that the -1 programmed ribosomal frameshifting mechanism in three of these genes (PEG10, PNMA3, and PNMA5) is conserved across mammals and originates alternative proteins. These genes were targets of positive selection in primates, and one of the positively selected sites affects a B-cell epitope on the spike domain of the PNMA5 capsid, a finding reminiscent of observations in infectious viruses. More generally, we found that retrotransposon-derived proteins vary in their intrinsically disordered region content and this is directly associated with their evolutionary rates. Most positively selected sites in these proteins are located in intrinsically disordered regions and some of them impact protein posttranslational modifications, such as autocleavage and phosphorylation. Detailed analyses of the biophysical properties of intrinsically disordered regions showed that positive selection preferentially targeted regions with lower conformational entropy. Furthermore, positive selection introduces variation in binary sequence patterns across orthologues, as well as in chain compaction. Our results shed light on the evolutionary trajectories of a unique class of mammalian genes and suggest a novel approach to study how intrinsically disordered region biophysical characteristics are affected by evolution.

Possible involvement of three-stemmed pseudoknots in regulating translational initiation in human mRNAs.

RNA pseudoknots play a crucial role in various cellular functions. Established pseudoknots show significant variation in both size and structural complexity. Specifically, three-stemmed pseudoknots are characterized by an additional stem-loop embedded in their structure. Recent findings highlight these pseudoknots as bacterial riboswitches and potent stimulators for programmed ribosomal frameshifting in RNA viruses like SARS-CoV2. To investigate the possible presence of functional three-stemmed pseudoknots in human mRNAs, we employed in-house developed computational methods to detect such structures within a dataset comprising 21,780 full-length human mRNA sequences. Numerous three-stemmed pseudoknots were identified. A selected set of 14 potential instances are presented, in which the start codon of the mRNA is found in close proximity either upstream, downstream, or within the identified three-stemmed pseudoknot. These pseudoknots likely play a role in translational initiation regulation. The probability of their existence gains support from their ranking as the most stable pseudoknot identified in the entire mRNA sequence, structural conservation across homologous mRNAs, stereochemical feasibility as demonstrated by structural modeling, and classification as members of the CPK-1 pseudoknot family, which includes many well-established pseudoknots. Furthermore, in four of the mRNAs, two or three closely spaced or tandem three-stemmed pseudoknots were identified. These findings suggest the frequent occurrence of three-stemmed pseudoknots in human mRNAs. A stepwise co-transcriptional folding mechanism is proposed for the formation of a three-stemmed pseudoknot structure. Our results not only provide fresh insights into the structures and functions of pseudoknots but also unveil the potential to target pseudoknots for treating human diseases.

The interferon-regulated host factor hnRNPA0 modulates HIV-1 production by interference with LTR activity, mRNA trafficking, and programmed ribosomal frameshifting.

The interplay between host factors and viral components impacts viral replication efficiency profoundly. Members of the cellular heterogeneous nuclear ribonucleoprotein family (hnRNPs) have been extensively studied as HIV-1 host dependency factors, but whether they play a role in innate immunity is currently unknown. This study aimed to identify hnRNPA0 as a type I interferon (IFN)-repressed host factor in HIV-1-infected cells. Knockdown of hnRNPA0, a situation that mirrors conditions under IFN stimulation, increased LTR activity, export of unspliced HIV-1 mRNA, viral particle production, and thus, increased infectivity. Conversely, hnRNPA0 overexpression primarily reduced plasmid-driven and integrated HIV-1 long terminal repeat (LTR) activity, significantly decreasing total viral mRNA and protein levels. In addition, high levels of hnRNPA0 significantly reduced the HIV-1 programmed ribosomal frameshifting efficiency, resulting in a shift in the HIV-1 p55/p15 ratio. The HIV-1 alternative splice site usage remained largely unaffected by altered hnRNPA0 levels suggesting that the synergistic inhibition of the LTR activity and viral mRNA transcription, as well as impaired ribosomal frameshifting efficiency, are critical factors for efficient HIV-1 replication regulated by hnRNPA0. The pleiotropic dose-dependent effects under high or low hnRNPA0 levels were further confirmed in HIV-1-infected Jurkat cells. Finally, our study revealed that hnRNPA0 levels in PBMCs were lower in therapy-naive HIV-1-infected individuals compared to healthy controls. Our findings highlight a significant role for hnRNPA0 in HIV-1 replication and suggest that its IFN-I-regulated expression levels are critical for viral fitness allowing replication in an antiviral environment.IMPORTANCERNA-binding proteins, in particular, heterogeneous nuclear ribonucleoproteins (hnRNPs), have been extensively studied. Some act as host dependency factors for HIV-1 since they are involved in multiple cellular gene expression processes. Our study revealed hnRNPA0 as an IFN-regulated host factor, that is differently expressed after IFN-I treatment in HIV-1 target cells and lower expressed in therapy-naïve HIV-1-infected individuals. Our findings demonstrate the significant pleiotropic role of hnRNPA0 in viral replication: In high concentrations, hnRNPA0 limits viral replication by negatively regulating Tat-LTR transcription, retaining unspliced mRNA in the nucleus, and significantly impairing programmed ribosomal frameshifting. Low hnRNPA0 levels as observed in IFN-treated THP-1 cells, particularly facilitate HIV LTR activity and unspliced mRNA export, suggesting a role in innate immunity in favor of HIV replication. Understanding the mode of action between hnRNPA0 and HIV-1 gene expression might help to identify novel therapeutically strategies against HIV-1 and other viruses.

Structure of the SARS-CoV-2 Frameshift Stimulatory Element with an Upstream Multibranch Loop.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshift stimulatory element (FSE) is necessary for programmed -1 ribosomal frameshifting (-1 PRF) and optimized viral efficacy. The FSE has an abundance of context-dependent alternate conformations, but two of the structures most crucial to -1 PRF are an attenuator hairpin and a three-stem H-type pseudoknot structure. A crystal structure of the pseudoknot alone features three RNA stems in a helically stacked linear structure, whereas a 6.9 Å cryo-EM structure including the upstream heptameric slippery site resulted in a bend between two stems. Our previous research alluded to an extended upstream multibranch loop that includes both the attenuator hairpin and the slippery site-a conformation not previously modeled. We aim to provide further context to the SARS-CoV-2 FSE via computational and medium resolution cryo-EM approaches, by presenting a 6.1 Å cryo-EM structure featuring a linear pseudoknot structure and a dynamic upstream multibranch loop.

Tying the knot: Unraveling the intricacies of the coronavirus frameshift pseudoknot.

Understanding and targeting functional RNA structures towards treatment of coronavirus infection can help us to prepare for novel variants of SARS-CoV-2 (the virus causing COVID-19), and any other coronaviruses that could emerge via human-to-human transmission or potential zoonotic (inter-species) events. Leveraging the fact that all coronaviruses use a mechanism known as -1 programmed ribosomal frameshifting (-1 PRF) to replicate, we apply algorithms to predict the most energetically favourable secondary structures (each nucleotide involved in at most one pairing) that may be involved in regulating the -1 PRF event in coronaviruses, especially SARS-CoV-2. We compute previously unknown most stable structure predictions for the frameshift site of coronaviruses via hierarchical folding, a biologically motivated framework where initial non-crossing structure folds first, followed by subsequent, possibly crossing (pseudoknotted), structures. Using mutual information from 181 coronavirus sequences, in conjunction with the algorithm KnotAli, we compute secondary structure predictions for the frameshift site of different coronaviruses. We then utilize the Shapify algorithm to obtain most stable SARS-CoV-2 secondary structure predictions guided by frameshift sequence-specific and genome-wide experimental data. We build on our previous secondary structure investigation of the singular SARS-CoV-2 68 nt frameshift element sequence, by using Shapify to obtain predictions for 132 extended sequences and including covariation information. Previous investigations have not applied hierarchical folding to extended length SARS-CoV-2 frameshift sequences. By doing so, we simulate the effects of ribosome interaction with the frameshift site, providing insight to biological function. We contribute in-depth discussion to contextualize secondary structure dual-graph motifs for SARS-CoV-2, highlighting the energetic stability of the previously identified 3_8 motif alongside the known dominant 3_3 and 3_6 (native-type) -1 PRF structures. Using a combination of thermodynamic methods and sequence covariation, our novel predictions suggest function of the attenuator hairpin via previously unknown pseudoknotted base pairing. While certain initial RNA folding is consistent, other pseudoknotted base pairs form which indicate potential conformational switching between the two structures.

Factor-Dependent Internal Ribosome Entry Site and -1 Programmed Frameshifting Signal in the Bemisia-Associated Dicistrovirus 2.

The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.

The Inhibition of Gag-Pol Expression by the Restriction Factor Shiftless Is Dispensable for the Restriction of HIV-1 Infection.

The interferon-induced host cell protein Shiftless (SFL) inhibits -1 programmed ribosomal frameshifting (-1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits -1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of -1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as -1PRF signals, both of which are regulated by mRNA secondary structure elements.

The application of single-molecule optical tweezers to study disease-related structural dynamics in RNA.

RNA, a dynamic and flexible molecule with intricate three-dimensional structures, has myriad functions in disease development. Traditional methods, such as X-ray crystallography and nuclear magnetic resonance, face limitations in capturing real-time, single-molecule dynamics crucial for understanding RNA function. This review explores the transformative potential of single-molecule force spectroscopy using optical tweezers, showcasing its capability to directly probe time-dependent structural rearrangements of individual RNA molecules. Optical tweezers offer versatility in exploring diverse conditions, with the potential to provide insights into how environmental changes, ligands and RNA-binding proteins impact RNA behaviour. By enabling real-time observations of large-scale structural dynamics, optical tweezers emerge as an invaluable tool for advancing our comprehension of RNA structure and function. Here, we showcase their application in elucidating the dynamics of RNA elements in virology, such as the pseudoknot governing ribosomal frameshifting in SARS-CoV-2.

Implication of nucleotides near the 3' end of 16S rRNA in guarding the translational reading frame.

Loss of the translational reading frame leads to misincorporation and premature termination, which can have lethal consequences. Based on structural evidence that A1503 of 16S rRNA intercalates between specific mRNA bases, we tested the possibility that it plays a role in maintenance of the reading frame by constructing ribosomes with an abasic nucleotide at position 1503. This was done by specific cleavage of 16S rRNA at position 1493 using the colicin E3 endonuclease and replacing the resulting 3'-terminal 49mer fragment with a synthetic oligonucleotide containing the abasic site using a novel splinted RNA ligation method. Ribosomes reconstituted from the abasic 1503 16S rRNA were highly active in protein synthesis but showed elevated levels of spontaneous frameshifting into the -1 reading frame. We then asked whether the residual frameshifting persisting in control ribosomes containing an intact A1503 is due to the absence of the N6-dimethyladenosine modifications at positions 1518 and 1519. Indeed, this frameshifting was rescued by site-specific methylation in vitro by the ksgA methylase. These findings thus implicate two different sites near the 3' end of 16S rRNA in maintenance of the translational reading frame, providing yet another example of a functional role for ribosomal RNA in protein synthesis.

PRFect: a tool to predict programmed ribosomal frameshifts in prokaryotic and viral genomes.

BACKGROUND: One of the stranger phenomena that can occur during gene translation is where, as a ribosome reads along the mRNA, various cellular and molecular properties contribute to stalling the ribosome on a slippery sequence and shifting the ribosome into one of the other two alternate reading frames. The alternate frame has different codons, so different amino acids are added to the peptide chain. More importantly, the original stop codon is no longer in-frame, so the ribosome can bypass the stop codon and continue to translate the codons past it. This produces a longer version of the protein, a fusion of the original in-frame amino acids, followed by all the alternate frame amino acids. There is currently no automated software to predict the occurrence of these programmed ribosomal frameshifts (PRF), and they are currently only identified by manual curation. RESULTS: Here we present PRFect, an innovative machine-learning method for the detection and prediction of PRFs in coding genes of various types. PRFect combines advanced machine learning techniques with the integration of multiple complex cellular properties, such as secondary structure, codon usage, ribosomal binding site interference, direction, and slippery site motif. Calculating and incorporating these diverse properties posed significant challenges, but through extensive research and development, we have achieved a user-friendly approach. The code for PRFect is freely available, open-source, and can be easily installed via a single command in the terminal. Our comprehensive evaluations on diverse organisms, including bacteria, archaea, and phages, demonstrate PRFect's strong performance, achieving high sensitivity, specificity, and an accuracy exceeding 90%. The code for PRFect is freely available and installs with a single terminal command. CONCLUSION: PRFect represents a significant advancement in the field of PRF detection and prediction, offering a powerful tool for researchers and scientists to unravel the intricacies of programmed ribosomal frameshifting in coding genes.

The Deficiency of Hypusinated eIF5A Decreases the Putrescine/Spermidine Ratio and Inhibits +1 Programmed Ribosomal Frameshifting during the Translation of Ty1 Retrotransposon in Saccharomyces cerevisiae.

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential in all eukaryotes. It is identified initially as an initiation factor and functions broadly in translation elongation and termination. The hypusination of eIF5A is specifically required for +1 PRF at the shifty site derived from the ornithine decarboxylase antizyme 1 (OAZ1) in Saccharomyces cerevisiae. However, whether the regulation of +1 PRF by yeast eIF5A is universal remains unknown. Here, we found that Sc-eIF5A depletion decreased the putrescine/spermidine ratio. The re-introduction of Sc-eIF5A in yeast eIF5A mutants recovered the putrescine/spermidine ratio. In addition, the Sc-eIF5A depletion decreases +1 PRF during the decoding of Ty1 retrotransposon mRNA, but has no effect on -1 PRF during the decoding of L-A virus mRNA. The re-introduction of Sc-eIF5A in yeast eIF5A mutants restored the +1 PRF rate of Ty1. The inhibition of the hypusine modification of yeast eIF5A by GC7 treatment or by mutating the hypusination site Lys to Arg caused decreases of +1 PRF rates in the Ty1 retrotransposon. Furthermore, mutational studies of the Ty1 frameshifting element support a model where the efficient removal of ribosomal subunits at the first Ty1 frame 0 stop codon is required for the frameshifting of trailing ribosomes. This dependency is likely due to the unique position of the frame 0 stop codon distance from the slippery sequence of Ty1. The results showed that eIF5A is a trans-regulator of +1 PRF for Ty1 retrotransposon and could function universally in yeast.

Systematic analysis of nonprogrammed frameshift suppression in E. coli via translational tiling proteomics.

The synthesis of proteins as encoded in the genome depends critically on translational fidelity. Nevertheless, errors inevitably occur, and those that result in reading frame shifts are particularly consequential because the resulting polypeptides are typically nonfunctional. Despite the generally maladaptive impact of such errors, the proper decoding of certain mRNAs, including many viral mRNAs, depends on a process known as programmed ribosomal frameshifting. The fact that these programmed events, commonly involving a shift to the -1 frame, occur at specific evolutionarily optimized "slippery" sites has facilitated mechanistic investigation. By contrast, less is known about the scope and nature of error (i.e., nonprogrammed) frameshifting. Here, we examine error frameshifting by monitoring spontaneous frameshift events that suppress the effects of single base pair deletions affecting two unrelated test proteins. To map the precise sites of frameshifting, we developed a targeted mass spectrometry-based method called "translational tiling proteomics" for interrogating the full set of possible -1 slippage events that could produce the observed frameshift suppression. Surprisingly, such events occur at many sites along the transcripts, involving up to one half of the available codons. Only a subset of these resembled canonical "slippery" sites, implicating alternative mechanisms potentially involving noncognate mispairing events. Additionally, the aggregate frequency of these events (ranging from 1 to 10% in our test cases) was higher than we might have anticipated. Our findings point to an unexpected degree of mechanistic diversity among ribosomal frameshifting events and suggest that frameshifted products may contribute more significantly to the proteome than generally assumed.

eIF5A promotes +1 programmed ribosomal frameshifting in Euplotes octocarinatus.

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The single-celled eukaryote Euplotes exhibit high frequency of PRF. However, the molecular mechanism of modulating Euplotes PRF remains largely unknown. Here, we identified two novel eIF5A genes, eIF5A1 and eIF5A2, in Euplotes octocarinatus and found that the Eo-eIF5A2 gene requires a -1 PRF to produce complete protein product. Although both Eo-eIF5As showed significant structural similarity with yeast eIF5A, neither of them could functionally replace yeast eIF5A. Eo-eIF5A knockdown inhibited +1 PRF of the η-tubulin gene. Using an in vitro reconstituted translation system, we found that hypusinated Eo-eIF5A (Eo-eIF5AH) can promote +1 PRF at the canonical AAA_UAA frameshifting site of Euplotes. The results showed eIF5A is a novel trans-regulator of PRF in Euplotes and has an evolutionary conserved role in regulating +1 PRF in eukaryotes.

High-throughput screening for a SARS-CoV-2 frameshifting inhibitor using a cell-free protein synthesis system.

Programmed-1 ribosomal frameshifting (-1 PRF) is a translational mechanism adopted by some viruses, including SARS-CoV-2. To find a compound that can inhibit -1 PRF in SARS-CoV-2, we set up a high-throughput screening system using a HeLa cell extract-derived cell-free protein synthesis (CFPS) system. A total of 32,000 compounds were individually incubated with the CFPS system programmed with a -1 PRF-EGFP template. Several compounds were observed to decrease the -1 PRF-driven fluorescence, and one of them had some suppressive effect on -1 PRF of a SARS-CoV-2 genome sequence in transfected cells. Thus the CFPS system can be used as a tool for a high-throughput screening of chemicals.

Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human.

Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.

Analysis of programmed frameshifting during translation of prfB in Flavobacterium johnsoniae.

Ribosomes of Bacteroidia fail to recognize Shine-Dalgarno (SD) sequences due to sequestration of the 3' tail of the 16S rRNA on the 30S platform. Yet in these organisms, the prfB gene typically contains the programmed +1 frameshift site with its characteristic SD sequence. Here, we investigate prfB autoregulation in Flavobacterium johnsoniae, a member of the Bacteroidia. We find that the efficiency of prfB frameshifting in F. johnsoniae is low (∼7%) relative to that in Escherichia coli (∼50%). Mutation or truncation of bS21 in F. johnsoniae increases frameshifting substantially, suggesting that anti-SD (ASD) sequestration is responsible for the reduced efficiency. The frameshift site of certain Flavobacteriales, such as Winogradskyella psychrotolerans, has no SD. In F. johnsoniae, this W. psychrotolerans sequence supports frameshifting as well as the native sequence, and mutation of bS21 causes no enhancement. These data suggest that prfB frameshifting normally occurs without SD-ASD pairing, at least under optimal laboratory growth conditions. Chromosomal mutations that remove the frameshift or ablate the SD confer subtle growth defects in the presence of paraquat or streptomycin, respectively, indicating that both the autoregulatory mechanism and the SD element contribute to F. johnsoniae cell fitness. Analysis of prfB frameshift sites across 2686 representative bacteria shows loss of the SD sequence in many clades, with no obvious relationship to genome-wide SD usage. These data reveal unexpected variation in the mechanism of frameshifting and identify another group of organisms, the Verrucomicrobiales, that globally lack SD sequences.

N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting.

In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1,2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3-5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization.

ORFeus: a computational method to detect programmed ribosomal frameshifts and other non-canonical translation events.

BACKGROUND: In canonical protein translation, ribosomes initiate translation at a specific start codon, maintain a single reading frame throughout elongation, and terminate at the first in-frame stop codon. However, ribosomal behavior can deviate at each of these steps, sometimes in a programmed manner. Certain mRNAs contain sequence and structural elements that cause ribosomes to begin translation at alternative start codons, shift reading frame, read through stop codons, or reinitiate on the same mRNA. These processes represent important translational control mechanisms that can allow an mRNA to encode multiple functional protein products or regulate protein expression. The prevalence of these events remains uncertain, due to the difficulty of systematic detection. RESULTS: We have developed a computational model to infer non-canonical translation events from ribosome profiling data. CONCLUSION: ORFeus identifies known examples of alternative open reading frames and recoding events across different organisms and enables transcriptome-wide searches for novel events.

Shiftless Is a Novel Member of the Ribosome Stress Surveillance Machinery That Has Evolved to Play a Role in Innate Immunity and Cancer Surveillance.

A longstanding paradox in molecular biology has centered on the question of how very long proteins are synthesized, despite numerous measurements indicating that ribosomes spontaneously shift reading frame at rates that should preclude their ability completely translate their mRNAs. Shiftless (SFL; C19orf66) was originally identified as an interferon responsive gene encoding an antiviral protein, indicating that it is part of the innate immune response. This activity is due to its ability to bind ribosomes that have been programmed by viral sequence elements to shift reading frame. Curiously, Shiftless is constitutively expressed at low levels in mammalian cells. This study examines the effects of altering Shiftless homeostasis, revealing how it may be used by higher eukaryotes to identify and remove spontaneously frameshifted ribosomes, resolving the apparent limitation on protein length. Data also indicate that Shiftless plays a novel role in the ribosome-associated quality control program. A model is proposed wherein SFL recognizes and arrests frameshifted ribosomes, and depending on SFL protein concentrations, either leads to removal of frameshifted ribosomes while leaving mRNAs intact, or to mRNA degradation. We propose that SFL be added to the growing pantheon of proteins involved in surveilling translational fidelity and controlling gene expression in higher eukaryotes.

Research Progress of Porcine Reproductive and Respiratory Syndrome Virus NSP2 Protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) is globally prevalent and seriously harms the economic efficiency of pig farming. Because of its immunosuppression and high incidence of mutant recombination, PRRSV poses a great challenge for disease prevention and control. Nonstructural protein 2 (NSP2) is the most variable functional protein in the PRRSV genome and can generate NSP2N and NSP2TF variants due to programmed ribosomal frameshifts. These variants are broad and complex in function and play key roles in numerous aspects of viral protein maturation, viral particle assembly, regulation of immunity, autophagy, apoptosis, cell cycle and cell morphology. In this paper, we review the structural composition, programmed ribosomal frameshift and biological properties of NSP2 to facilitate basic research on PRRSV and to provide theoretical support for disease prevention and control and therapeutic drug development.