Programmed ribosomal frameshifting during PLEKHM2 mRNA decoding generates a constitutively active mediator of kinesin-1-dependent lysosome transport.

Programmed ribosomal frameshifting is a translational recoding phenomenon in which a proportion of ribosomes are stimulated to slip backwards or forwards on an mRNA1, rephasing the ribosome relative to the mRNA. While frameshifting is often employed by viruses2, very few phylogenetically conserved examples are known in vertebrate genes and the evidence for some of these is controversial3,4. Here we report a +1 frameshifting signal in the coding sequence of the human gene PLEKHM2, encoding the ARL8-dependent, lysosome-kinesin-1 adaptor protein PLEKHM25. This +1 frameshifting signal, UCC_UUU_CGG, is highly conserved in vertebrates and exhibits an influenza virus-like frameshift motif with similar efficiency6,7. Purification and mass spectrometry of GFP-tagged trans-frame protein from cells confirms frameshifting. Structure prediction shows that the new C-terminal domain generated by this frameshift forms an alpha-helix. This additional domain relieves PLEKHM2 from autoinhibition, allowing it to move to the tips of cells via association with kinesin-1 without requiring activation by ARL8. Thus, the frameshift proteoform generates a constitutively active adaptor of kinesin-1.

Streptomyces rare codon UUA: from features associated with 2 adpA related locations to candidate phage regulatory translational bypassing.

In Streptomyces species, the cell cycle involves a switch from an early and vegetative state to a later phase where secondary products including antibiotics are synthesized, aerial hyphae form and sporulation occurs. AdpA, which has two domains, activates the expression of numerous genes involved in the switch from the vegetative growth phase. The adpA mRNA of many Streptomyces species has a UUA codon in a linker region between 5' sequence encoding one domain and 3' sequence encoding its other and C-terminal domain. UUA codons are exceptionally rare in Streptomyces, and its functional cognate tRNA is not present in a fully modified and acylated form, in the early and vegetative phase of the cell cycle though it is aminoacylated later. Here, we report candidate recoding signals that may influence decoding of the linker region UUA. Additionally, a short ORF 5' of the main ORF has been identified with a GUG at, or near, its 5' end and an in-frame UUA near its 3' end. The latter is commonly 5 nucleotides 5' of the main ORF start. Ribosome profiling data show translation of that 5' region. Ten years ago, UUA-mediated translational bypassing was proposed as a sensor by a Streptomyces phage of its host's cell cycle stage and an effector of its lytic/lysogeny switch. We provide the first experimental evidence supportive of this proposal.

Our current understanding of the toxicity of altered mito-ribosomal fidelity during mitochondrial protein synthesis: What can it tell us about human disease?

Altered mito-ribosomal fidelity is an important and insufficiently understood causative agent of mitochondrial dysfunction. Its pathogenic effects are particularly well-known in the case of mitochondrially induced deafness, due to the existence of the, so called, ototoxic variants at positions 847C (m.1494C) and 908A (m.1555A) of 12S mitochondrial (mt-) rRNA. It was shown long ago that the deleterious effects of these variants could remain dormant until an external stimulus triggered their pathogenicity. Yet, the link from the fidelity defect at the mito-ribosomal level to its phenotypic manifestation remained obscure. Recent work with fidelity-impaired mito-ribosomes, carrying error-prone and hyper-accurate mutations in mito-ribosomal proteins, have started to reveal the complexities of the phenotypic manifestation of mito-ribosomal fidelity defects, leading to a new understanding of mtDNA disease. While much needs to be done to arrive to a clear picture of how defects at the level of mito-ribosomal translation eventually result in the complex patterns of disease observed in patients, the current evidence indicates that altered mito-ribosome function, even at very low levels, may become highly pathogenic. The aims of this review are three-fold. First, we compare the molecular details associated with mito-ribosomal fidelity to those of general ribosomal fidelity. Second, we gather information on the cellular and organismal phenotypes associated with defective translational fidelity in order to provide the necessary grounds for an understanding of the phenotypic manifestation of defective mito-ribosomal fidelity. Finally, the results of recent experiments directly tackling mito-ribosomal fidelity are reviewed and future paths of investigation are discussed.

Nontriplet feature of genetic code in Euplotes ciliates is a result of neutral evolution.

The triplet nature of the genetic code is considered a universal feature of known organisms. However, frequent stop codons at internal mRNA positions in Euplotes ciliates ultimately specify ribosomal frameshifting by one or two nucleotides depending on the context, thus posing a nontriplet feature of the genetic code of these organisms. Here, we sequenced transcriptomes of eight Euplotes species and assessed evolutionary patterns arising at frameshift sites. We show that frameshift sites are currently accumulating more rapidly by genetic drift than they are removed by weak selection. The time needed to reach the mutational equilibrium is several times longer than the age of Euplotes and is expected to occur after a several-fold increase in the frequency of frameshift sites. This suggests that Euplotes are at an early stage of the spread of frameshifting in expression of their genome. In addition, we find the net fitness burden of frameshift sites to be noncritical for the survival of Euplotes. Our results suggest that fundamental genome-wide changes such as a violation of the triplet character of genetic code can be introduced and maintained solely by neutral evolution.

The translating bacterial ribosome at 1.55 Å resolution generated by cryo-EM imaging services.

Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome. This complex macromolecular machine is the target of important antimicrobial drugs, an integral line of defence against infectious diseases. Here, we describe how open access to cryo-electron microscopy facilities combined with bespoke user support enabled structural determination of the translating ribosome from Escherichia coli at 1.55 Å resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enable interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 Å resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.

Monitoring translation in all reading frames downstream of weak stop codons provides mechanistic insights into the impact of nucleotide and cellular contexts.

A stop codon entering the ribosome A-site is normally decoded by release factors that induce release of the polypeptide. Certain factors influence the efficiency of the termination which is in competition with elongation in either the same (readthrough) or an alternative (frameshifting) reading frame. To gain insight into the competition between these processes, we monitored translation in parallel from all three reading frames downstream of stop codons while changing the nucleotide context of termination sites or altering cellular conditions (polyamine levels). We found that P-site codon identity can have a major impact on the termination efficiency of the OPRL1 stop signal, whereas for the OAZ1 ORF1 stop signal, the P-site codon mainly influences the reading frame of non-terminating ribosomes. Changes to polyamine levels predominantly influence the termination efficiency of the OAZ1 ORF1 stop signal. In contrast, increasing polyamine levels stimulate readthrough of the OPRL1 stop signal by enhancing near-cognate decoding rather than by decreasing termination efficiency. Thus, by monitoring the four competing processes occurring at stop codons we were able to determine which is the most significantly affected upon perturbation. This approach may be useful for the interrogation of other recoding phenomena where alternative decoding processes compete with standard decoding.

Lack of evidence for ribosomal frameshifting in ATP7B mRNA decoding.

The research article describing the discovery of ribosomal frameshifting in the bacterial CopA gene also reported the occurrence of frameshifting in the expression of the human ortholog ATP7B based on assays using dual luciferase reporters. An examination of the publicly available ribosome profiling data and the phylogenetic analysis of the proposed frameshifting site cast doubt on the validity of this claim and prompted us to reexamine the evidence. We observed similar apparent frameshifting efficiencies as the original authors using the same type of vector that synthesizes both luciferases as a single polyprotein. However, we noticed anomalously low absolute luciferase activities from the N-terminal reporter that suggests interference of reporter activity or levels by the ATP7B test cassette. When we tested the same proposed ATP7B frameshifting cassette in a more recently developed reporter system in which the reporters are released without being included in a polyprotein, no frameshifting was detected above background levels.

Maximum depth sequencing reveals an ON/OFF replication slippage switch and apparent in vivo selection for bifidobacterial pilus expression.

The human gut microbiome, of which the genus Bifidobacterium is a prevalent and abundant member, is thought to sustain and enhance human health. Several surface-exposed structures, including so-called sortase-dependent pili, represent important bifidobacterial gut colonization factors. Here we show that expression of two sortase-dependent pilus clusters of the prototype Bifidobacterium breve UCC2003 depends on replication slippage at an intragenic G-tract, equivalents of which are present in various members of the Bifidobacterium genus. The nature and extent of this slippage is modulated by the host environment. Involvement of such sortase-dependent pilus clusters in microbe-host interactions, including bacterial attachment to the gut epithelial cells, has been shown previously and is corroborated here for one case. Using a Maximum Depth Sequencing strategy aimed at excluding PCR and sequencing errors introduced by DNA polymerase reagents, specific G-tract sequences in B. breve UCC2003 reveal a range of G-tract lengths whose plasticity within the population is functionally utilized. Interestingly, replication slippage is shown to be modulated under in vivo conditions in a murine model. This in vivo modulation causes an enrichment of a G-tract length which appears to allow biosynthesis of these sortase-dependent pili. This work provides the first example of productive replication slippage influenced by in vivo conditions. It highlights the potential for microdiversity generation in "beneficial" gut commensals.

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.

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome.

Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.

Tissue-specific dynamic codon redefinition in Drosophila.

Translational stop codon readthrough occurs in organisms ranging from viruses to mammals and is especially prevalent in decoding Drosophila and viral mRNAs. Recoding of UGA, UAG, or UAA to specify an amino acid allows a proportion of the protein encoded by a single gene to be C-terminally extended. The extended product from Drosophila kelch mRNA is 160 kDa, whereas unextended Kelch protein, a subunit of a Cullin3-RING ubiquitin ligase, is 76 kDa. Previously we reported tissue-specific regulation of readthrough of the first kelch stop codon. Here, we characterize major efficiency differences in a variety of cell types. Immunoblotting revealed low levels of readthrough in malpighian tubules, ovary, and testis but abundant readthrough product in lysates of larval and adult central nervous system (CNS) tissue. Reporters of readthrough demonstrated greater than 30% readthrough in adult brains, and imaging in larval and adult brains showed that readthrough occurred in neurons but not glia. The extent of readthrough stimulatory sequences flanking the readthrough stop codon was assessed in transgenic Drosophila and in human tissue culture cells where inefficient readthrough occurs. A 99-nucleotide sequence with potential to form an mRNA stem-loop 3' of the readthrough stop codon stimulated readthrough efficiency. However, even with just six nucleotides of kelch mRNA sequence 3' of the stop codon, readthrough efficiency only dropped to 6% in adult neurons. Finally, we show that high-efficiency readthrough in the Drosophila CNS is common; for many neuronal proteins, C-terminal extended forms of individual proteins are likely relatively abundant.

Unusually efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF.

While near-cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here, we describe a rare case of highly efficient near-cognate initiation. A CUG triplet located in the 5' leader of POLG messenger RNA (mRNA) initiates almost as efficiently (∼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF), which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5' of this CUG governs the ratio between POLG (the catalytic subunit of mitochondrial DNA polymerase) and POLGARF synthesized from a single POLG mRNA. Functional investigation of POLGARF suggests a role in extracellular signaling. While unprocessed POLGARF localizes to the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid cleavage and secretion of a POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ∼160 million y ago due to a mammalian-wide interspersed repeat (MIR) transposition into the 5' leader sequence of the mammalian POLG gene, which became fixed in placental mammals. This discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution.

Polysomes Bypass a 50-Nucleotide Coding Gap Less Efficiently Than Monosomes Due to Attenuation of a 5' mRNA Stem-Loop and Enhanced Drop-off.

Efficient translational bypassing of a 50-nt non-coding gap in a phage T4 topoisomerase subunit gene (gp60) requires several recoding signals. Here we investigate the function of the mRNA stem-loop 5' of the take-off codon, as well as the importance of ribosome loading density on the mRNA for efficient bypassing. We show that polysomes are less efficient at mediating bypassing than monosomes, both in vitro and in vivo, due to their preventing formation of a stem-loop 5' of the take-off codon and allowing greater peptidyl-tRNA drop off. A ribosome profiling analysis of phage T4-infected Escherichia coli yielded protected mRNA fragments within the normal size range derived from ribosomes stalled at the take-off codon. However, ribosomes at this position also yielded some 53-nucleotide fragments, 16 longer. These were due to protection of the nucleotides that form the 5' stem-loop. NMR shows that the 5' stem-loop is highly dynamic. The importance of different nucleotides in the 5' stem-loop is revealed by mutagenesis studies. These data highlight the significance of the 5' stem-loop for the 50-nt bypassing and further enhance appreciation of relevance of the extent of ribosome loading for recoding.

The energy landscape of -1 ribosomal frameshifting.

Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3' messenger RNA (mRNA) structures, but how these mRNA structures enhance -1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal -1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.

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.

Processive Recoding and Metazoan Evolution of Selenoprotein P: Up to 132 UGAs in Molluscs.

Selenoproteins typically contain a single selenocysteine, the 21st amino acid, encoded by a context-redefined UGA. However, human selenoprotein P (SelenoP) has a redox-functioning selenocysteine in its N-terminal domain and nine selenium transporter-functioning selenocysteines in its C-terminal domain. Here we show that diverse SelenoP genes are present across metazoa with highly variable numbers of Sec-UGAs, ranging from a single UGA in certain insects, to 9 in common spider, and up to 132 in bivalve molluscs. SelenoP genes were shaped by a dynamic evolutionary process linked to selenium usage. Gene evolution featured modular expansions of an ancestral multi-Sec domain, which led to particularly Sec-rich SelenoP proteins in many aquatic organisms. We focused on molluscs, and chose Pacific oyster Magallana gigas as experimental model. We show that oyster SelenoP mRNA with 46 UGAs is translated full-length in vivo. Ribosome profiling indicates that selenocysteine specification occurs with ∼5% efficiency at UGA1 and approaches 100% efficiency at distal 3' UGAs. We report genetic elements relevant to its expression, including a leader open reading frame and an RNA structure overlapping the initiation codon that modulates ribosome progression in a selenium-dependent manner. Unlike their mammalian counterparts, the two SECIS elements in oyster SelenoP (3'UTR recoding elements) do not show functional differentiation in vitro. Oysters can increase their tissue selenium level up to 50-fold upon supplementation, which also results in extensive changes in selenoprotein expression.