The Mytilus chilensis Steamer-like Element-1 Retrotransposon Antisense mRNA Harbors an Internal Ribosome Entry Site That Is Modulated by hnRNPK.

LTR-retrotransposons are transposable elements characterized by the presence of long terminal repeats (LTRs) directly flanking an internal coding region. They share genome organization and replication strategies with retroviruses. Steamer-like Element-1 (MchSLE-1) is an LTR-retrotransposon identified in the genome of the Chilean blue mussel Mytilus chilensis. MchSLE-1 is transcribed; however, whether its RNA is also translated and the mechanism underlying such translation remain to be elucidated. Here, we characterize the MchSLE-1 translation mechanism. We found that the MchSLE-1 5' and 3'LTRs command transcription of sense and antisense RNAs, respectively. Using luciferase reporters commanded by the untranslated regions (UTRs) of MchSLE-1, we found that in vitro 5'UTR sense is unable to initiate translation, whereas the antisense 5'UTR initiates translation even when the eIF4E-eIF4G interaction was disrupted, suggesting the presence of an internal ribosomal entry site (IRES). The antisense 5'UTR IRES activity was tested using bicistronic reporters. The antisense 5'UTR has IRES activity only when the mRNA is transcribed in the nucleus, suggesting that nuclear RNA-binding proteins are required to modulate its activity. Indeed, heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as an IRES trans-acting factor (ITAF) of the MchSLE-1 IRES. To our knowledge, this is the first report describing an IRES in an antisense mRNA derived from a mussel LTR-retrotransposon.

Mitochondrial Ribosomal Protein MRPS15 Is a Component of Cytosolic Ribosomes and Regulates Translation in Stressed Cardiomyocytes.

Regulation of mRNA translation is a crucial step in controlling gene expression in stressed cells, impacting many pathologies, including heart ischemia. In recent years, ribosome heterogeneity has emerged as a key control mechanism driving the translation of subsets of mRNAs. In this study, we investigated variations in ribosome composition in human cardiomyocytes subjected to endoplasmic reticulum stress induced by tunicamycin treatment. Our findings demonstrate that this stress inhibits global translation in cardiomyocytes while activating internal ribosome entry site (IRES)-dependent translation. Analysis of translating ribosome composition in stressed and unstressed cardiomyocytes was conducted using mass spectrometry. We observed no significant changes in ribosomal protein composition, but several mitochondrial ribosomal proteins (MRPs) were identified in cytosolic polysomes, showing drastic variations between stressed and unstressed cells. The most notable increase in polysomes of stressed cells was observed in MRPS15. Its interaction with ribosomal proteins was confirmed by proximity ligation assay (PLA) and immunoprecipitation, suggesting its intrinsic role as a ribosomal component during stress. Knock-down or overexpression experiments of MRPS15 revealed its role as an activator of IRES-dependent translation. Furthermore, polysome profiling after immunoprecipitation with anti-MRPS15 antibody revealed that the "MRPS15 ribosome" is specialized in translating mRNAs involved in the unfolded protein response.

A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation.

Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology. IMPORTANCE: The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.

Nuclear ribonucleoprotein RALY downregulates foot-and-mouth disease virus replication but antagonized by viral 3C protease.

The internal ribosome entry site (IRES) element constitutes a cis-acting RNA regulatory sequence that recruits the ribosomal initiation complex in a cap-independent manner, assisted by various RNA-binding proteins and IRES trans-acting factors. Foot-and-mouth disease virus (FMDV) contains a functional IRES element and takes advantage of this element to subvert host translation machinery. Our study identified a novel mechanism wherein RALY, a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family belonging to RNA-binding proteins, binds to the domain 3 of FMDV IRES via its RNA recognition motif residue. This interaction results in the downregulation of FMDV replication by inhibiting IRES-driven translation. Furthermore, our findings reveal that the inhibitory effect exerted by RALY on FMDV replication is not attributed to the FMDV IRES-mediated assembly of translation initiation complexes but rather to the impediment of 80S ribosome complex formation after binding with 40S ribosomes. Conversely, 3Cpro of FMDV counteracts RALY-mediated inhibition by the ubiquitin-proteasome pathway. Therefore, these results indicate that RALY, as a novel critical IRES-binding protein, inhibits FMDV replication by blocking the formation of 80S ribosome, providing a deeper understanding of how viruses recruit and manipulate host factors. IMPORTANCE: The translation of FMDV genomic RNA driven by IRES element is a crucial step for virus infections. Many host proteins are hijacked to regulate FMDV IRES-dependent translation, but the regulatory mechanism remains unknown. Here, we report for the first time that cellular RALY specifically interacts with the IRES of FMDV and negatively regulates viral replication by blocking 80S ribosome assembly on FMDV IRES. Conversely, RALY-mediated inhibition is antagonized by the viral 3C protease by the ubiquitin-proteasome pathway. These results would facilitate further understanding of virus-host interactions and translational control during viral infection.

Heterogeneous nuclear ribonucleoprotein K promotes cap-independent translation initiation of retroviral mRNAs.

Translation initiation of the human immunodeficiency virus-type 1 (HIV-1) genomic mRNA (vRNA) is cap-dependent or mediated by an internal ribosome entry site (IRES). The HIV-1 IRES requires IRES-transacting factors (ITAFs) for function. In this study, we evaluated the role of the heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a potential ITAF for the HIV-1 IRES. In HIV-1-expressing cells, the depletion of hnRNPK reduced HIV-1 vRNA translation. Furthermore, both the depletion and overexpression of hnRNPK modulated HIV-1 IRES activity. Phosphorylations and protein arginine methyltransferase 1 (PRMT1)-induced asymmetrical dimethylation (aDMA) of hnRNPK strongly impacted the protein's ability to promote the activity of the HIV-1 IRES. We also show that hnRNPK acts as an ITAF for the human T cell lymphotropic virus-type 1 (HTLV-1) IRES, present in the 5'UTR of the viral sense mRNA, but not for the IRES present in the antisense spliced transcript encoding the HTLV-1 basic leucine zipper protein (sHBZ). This study provides evidence for a novel role of the host hnRNPK as an ITAF that stimulates IRES-mediated translation initiation for the retroviruses HIV-1 and HTLV-1.

Variable eIF4E-binding sites and their synergistic effect on cap-independent translation in a novel IRES of wheat yellow mosaic virus RNA2 isolates.

Some viral proteins are translated cap-independently via the internal ribosome entry site (IRES), which maintains conservative characteristic among different isolates of the same virus species. However, IRES activity showed a 7-fold variance in RNA2 of wheat yellow mosaic virus (WYMV) HC and LYJN isolates in this study. Based on RNA structure probing and mutagenesis assay, the loosened middle stem of H1 and the hepta-nucleotide top loop of H2 in the LYJN isolate synergistically ensured higher IRES activity than that in the HC isolate. In addition, the conserved top loop of H1 ensured basic IRES activity in HC and LYJN isolates. WYMV RNA2 5'-UTR specifically interacted with the wheat eIF4E, accomplished by the top loop of H1 in the HC isolate or the top loop of H1 and H2 in the LYJN isolate. The high IRES activity of the WYMV RNA2 LYJN isolate was regulated by two eIF4E-binding sites, which showed a synergistic effect mediated by the proximity of the H1 and H2 top loops owing to the flexibility of the middle stem in H1. This report presents a novel evolution pattern of IRES, which altered the number of eIF4E-binding sites to regulate IRES activity.

Validating the EMCV IRES Secondary Structure with Structure-Function Analysis.

The encephalomyocarditis virus internal ribosome entry site (EMCV IRES) is a structured RNA sequence found in the 5' UTR of the genomic RNA of the encephalomyocarditis virus. The EMCV IRES structure facilitates efficient translation initiation without needing a 5' m7G cap or the cap-binding protein eIF4E. The secondary structure of IRES has been the subject of several previous studies, and a number of different structural models have been proposed. Though some domains of the IRES are conserved across the different secondary structure models, domain I of the IRES varies greatly across them. A literature comparison led to the identification of three regions of interest that display structural heterogeneity within past secondary structure models. To test the accuracy of the secondary structure models in these regions, we employed mutational analysis and SHAPE probing. Mutational analysis revealed that two helical regions within the identified regions of interest are important for IRES translation. These helical regions are consistent with only one of the structure predictions in the literature and do not form in EMCV IRES structures predicted using modern secondary structure prediction methods. The importance of these regions is further supported by multiple SHAPE protections when probing was performed after in vitro translation, indicating that these regions are involved in the IRES translation complex. This work validates a published structure and demonstrates the importance of domain I during EMCV IRES translation initiation.

Rational design of microRNA-responsive switch for programmable translational control in mammalian cells.

Artificial RNA translation modulation usually relies on multiple components, such as RNA binding proteins (RBPs) or microRNAs (miRNAs) for off-switches and double-inverter cascades for on-switches. Recently, translational circular RNAs (circRNAs) were developed as promising alternatives for linear messenger RNAs (mRNAs). However, circRNAs still lack straightforward and programmable translation control strategies. Here, we rationally design a programmable miRNA-responsive internal ribosome entry site (IRES) translation activation and repression (PROMITAR) platform capable of implementing miRNA-based translation upregulation and downregulation in a single RNA construct. Based on the PROMITAR platform, we construct logic gates and cell-type classifier circRNAs and successfully identify desired mammalian cell types. We also demonstrate the potential therapeutic application of our platform for targeted cancer cell killing by encoding a cytotoxic protein in our engineered circRNAs. We expect our platform to expand the toolbox for RNA synthetic biology and provide an approach for potential biomedical applications in the future.

RNA-based translation activators for targeted gene upregulation.

Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present "translation-activating RNAs" (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research.

The intrinsically disordered region of eIF5B stimulates IRES usage and nucleates biological granule formation.

Cells activate stress response pathways to survive adverse conditions. Such responses involve the inhibition of global cap-dependent translation. This inhibition is a block that essential transcripts must escape via alternative methods of translation initiation, e.g., an internal ribosome entry site (IRES). IRESs have distinct structures and generally require a limited repertoire of translation factors. Cellular IRESs have been identified in many critical cellular stress response transcripts. We previously identified cellular IRESs in the murine insulin receptor (Insr) and insulin-like growth factor 1 receptor (Igf1r) transcripts and demonstrated their resistance to eukaryotic initiation factor 4F (eIF4F) inhibition. Here, we find that eIF5B preferentially promotes Insr, Igf1r, and hepatitis C virus IRES activity through a non-canonical mechanism that requires its highly charged and disordered N terminus. We find that the N-terminal region of eIF5B can drive cytoplasmic granule formation. This eIF5B granule is triggered by cellular stress and is sufficient to specifically promote IRES activity.

Translation of paired box 6 (PAX6) mRNA is IRES-mediated and inhibited by cymarin in breast cancer cells.

Paired box 6 (PAX6) is a member of the PAX family and plays an essential role in cancer cell cycle progression, colony formation, proliferation and invasion. Its expression is upregulated in many cancers including breast cancer, but the process of PAX6 mRNA translation has rarely been studied. We found that PAX6 translation level increased in MCF-7 breast cancer cells treated with the chemotherapeutic drug adriamycin (ADM), which might be attributable to internal ribosome entry site (IRES)-mediated translation. By modifying a bicistronic luciferase plasmid that is widely used to examine IRES activity, we found that the 469-base 5'-UTR of PAX6 mRNA contains an IRES element and that core IRES activity is located between nucleotides 159 and 333. Moreover, PAX6 IRES activity was induced during ADM treatment, which may be the main reason for the elevated level of PAX6 protein. We also found that cymarin, a cardiac glycoside, acts as an inhibitor of PAX6 protein expression by impairing its IRES-mediated translation. Furthermore, MCF-7 cell proliferation was suppressed during treatment with cymarin. These results provide novel insights into the translation mechanism of PAX6 in breast cancer cells and suggest that cymarin is a promising candidate for the treatment of breast cancer via targeting the expression of PAX6.

Integrative solution structure of PTBP1-IRES complex reveals strong compaction and ordering with residual conformational flexibility.

RNA-binding proteins (RBPs) are crucial regulators of gene expression, often composed of defined domains interspersed with flexible, intrinsically disordered regions. Determining the structure of ribonucleoprotein (RNP) complexes involving such RBPs necessitates integrative structural modeling due to their lack of a single stable state. In this study, we integrate magnetic resonance, mass spectrometry, and small-angle scattering data to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1/hnRNP I) bound to an RNA fragment from the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). This binding, essential for enhancing the translation of viral RNA, leads to a complex structure that demonstrates RNA and protein compaction, while maintaining pronounced conformational flexibility. Acting as an RNA chaperone, PTBP1 orchestrates the IRES RNA into a few distinct conformations, exposing the RNA stems outward. This conformational diversity is likely common among RNP structures and functionally important. Our approach enables atomic-level characterization of heterogeneous RNP structures.

Antibody designing against IIIabc junction (JIIIabc) of HCV IRES through affinity maturation; RNA-Antibody docking and interaction analysis.

Hepatitis C virus is a single-stranded RNA based virus which can cause chronic HCV and hepatocellular carcinoma. HCV genotype 3a has relatively higher rate of fibrosis progression, prevalence of steatosis and incidence of HCC. Despite HCVs variation in genomic sequence, the 5' untranslated region containing internal ribosome entry site (IRES) is highly conserved among all genotypes. It is responsible for translation and initiation of the viral protein. In present study, IRES was targeted by designing variants of reported antigen binding fragment (Fab) through affinity maturation approach. Affinity maturation strategy allowed the rational antibody designing with better biophysical properties and antibody-antigen binding interactions. Complementarity determining regions of reported Fab (wild type) were assessed and docked with IRES. Best generated model of Fab was selected and subjected to alanine scanning Three sets of insilico mutations for variants (V) designing were selected; single (1-71), double (a-j) and triple (I-X). Redocking of IRES-Fab variants consequently enabled the discovery of three variants exhibiting better docking score as compared to the wild type Fab. V1, V39 and V4 exhibited docking scores of -446.51, -446.52 and-446.29 kcal/mol respectively which is better as compared to the wild type Fab that exhibited the docking score of -351.23 kcal/mol. Variants exhibiting better docking score were screened for aggregation propensity by assessing the aggregation prone regions in Fab structure. Total A3D scores of wild type Fab, V1, V4 and V39 were predicted as -315.325, -312.727, -316.967 and -317.545 respectively. It is manifested that solubility of V4 and V39 is comparable to wild type Fab. In future, development and invitro assessment of these promising Fab HCV3 variants is aimed.

Rational design of eukaryotic riboswitches that up-regulate IRES-mediated translation initiation with high switching efficiency through a kinetic trapping mechanism in vitro.

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

Increased levels of eIF2A inhibit translation by sequestering 40S ribosomal subunits.

eIF2A was the first eukaryotic initiator tRNA carrier discovered but its exact function has remained enigmatic. Uncharacteristic of translation initiation factors, eIF2A is reported to be non-cytosolic in multiple human cancer cell lines. Attempts to study eIF2A mechanistically have been limited by the inability to achieve high yield of soluble recombinant protein. Here, we developed a purification paradigm that yields ∼360-fold and ∼6000-fold more recombinant human eIF2A from Escherichia coli and insect cells, respectively, than previous reports. Using a mammalian in vitro translation system, we found that increased levels of recombinant human eIF2A inhibit translation of multiple reporter mRNAs, including those that are translated by cognate and near-cognate start codons, and does so prior to start codon recognition. eIF2A also inhibited translation directed by all four types of cap-independent viral IRESs, including the CrPV IGR IRES that does not require initiation factors or initiator tRNA, suggesting excess eIF2A sequesters 40S subunits. Supplementation with additional 40S subunits prevented eIF2A-mediated inhibition and pull-down assays demonstrated direct binding between recombinant eIF2A and purified 40S subunits. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering 40S ribosomal subunits.

Dynamically regulated two-site interaction of viral RNA to capture host translation initiation factor.

Many RNA viruses employ internal ribosome entry sites (IRESs) in their genomic RNA to commandeer the host's translational machinery for replication. The IRES from encephalomyocarditis virus (EMCV) interacts with eukaryotic translation initiation factor 4 G (eIF4G), recruiting the ribosomal subunit for translation. Here, we analyze the three-dimensional structure of the complex composed of EMCV IRES, the HEAT1 domain fragment of eIF4G, and eIF4A, by cryo-electron microscopy. Two distinct eIF4G-interacting domains on the IRES are identified, and complex formation changes the angle therebetween. Further, we explore the dynamics of these domains by using solution NMR spectroscopy, revealing conformational equilibria in the microsecond to millisecond timescale. In the lowly-populated conformations, the base-pairing register of one domain is shifted with the structural transition of the three-way junction, as in the complex structure. Our study provides insights into the viral RNA's sophisticated strategy for optimal docking to hijack the host protein.

RB1 5́UTR contains an IRES related to cell cycle control and cancer progression.

Retinoblastoma gene1 (RB1) is the first tumor suppressor gene that stands as the guardian of the gate of the G1 period and plays a central role in proliferation and differentiation. However, no reports focused on the possible internal ribosome entry site (IRES) function of the RB1 gene flanking sequence. In this study, we constructed a bicistronic reporter with the RB1 5'untranslated region (5́UTR) inserted between two reporter coding regions. We found RB1 5'UTR harbors an IRES and has higher activity in cancer cell lines than normal cells. Besides, RB1 IRES acquired the highest activity in the G0/G1 phase of the cell cycle, and the RB1 5'UTR mutation collected from retinoblastoma decreased IRES activity compared with RB1 5'UTR wild-type. These data indicated that RB1 IRES is a mechanism of stress regulation and is related to cell cycle control and cancer progression.

DeepCIP: A multimodal deep learning method for the prediction of internal ribosome entry sites of circRNAs.

Circular RNAs (circRNAs) have been found to have the ability to encode proteins through internal ribosome entry sites (IRESs), which are essential RNA regulatory elements for cap-independent translation. Identification of IRES elements in circRNA is crucial for understanding its function. Previous studies have presented IRES predictors based on machine learning techniques, but they were mainly designed for linear RNA IRES. In this study, we proposed DeepCIP (Deep learning method for CircRNA IRES Prediction), a multimodal deep learning approach that employs both sequence and structural information for circRNA IRES prediction. Our results demonstrate the effectiveness of the sequence and structure models used by DeepCIP in feature extraction and suggest that integrating sequence and structural information efficiently improves the accuracy of prediction. The comparison studies indicate that DeepCIP outperforms other comparative methods on the test set and real circRNA IRES dataset. Furthermore, through the integration of an interpretable analysis mechanism, we elucidate the sequence patterns learned by our model, which align with the previous discovery of motifs that facilitate circRNA translation. Thus, DeepCIP has the potential to enhance the study of the coding potential of circRNAs and contribute to the design of circRNA-based drugs. DeepCIP as a standalone program is freely available at https://github.org/zjupgx/DeepCIP.

The structure and mechanism of action of a distinct class of dicistrovirus intergenic region IRESs.

Internal ribosomal entry sites (IRESs) engage with the eukaryotic translation apparatus to promote end-independent initiation. We identified a conserved class of ∼150 nt long intergenic region (IGR) IRESs in dicistrovirus genomes derived from members of the phyla Arthropoda, Bryozoa, Cnidaria, Echinodermata, Entoprocta, Mollusca and Porifera. These IRESs, exemplified by Wenling picorna-like virus 2, resemble the canonical cricket paralysis virus (CrPV) IGR IRES in comprising two nested pseudoknots (PKII/PKIII) and a 3'-terminal pseudoknot (PKI) that mimics a tRNA anticodon stem-loop base-paired to mRNA. However, they are ∼50 nt shorter than CrPV-like IRESs, and PKIII is an H-type pseudoknot that lacks the SLIV and SLV stem-loops that are primarily responsible for the affinity of CrPV-like IRESs for the 40S ribosomal subunit and that restrict initial binding of PKI to its aminoacyl (A) site. Wenling-class IRESs bound strongly to 80S ribosomes but only weakly to 40S subunits. Whereas CrPV-like IRESs must be translocated from the A site to the peptidyl (P) site by elongation factor 2 for elongation to commence, Wenling-class IRESs bound directly to the P site of 80S ribosomes, and decoding begins without a prior translocation step. A chimeric CrPV clone containing a Wenling-class IRES was infectious, confirming that the IRES functioned in cells.

Sordarin bound eEF2 unlocks spontaneous forward and reverse translocation on CrPV IRES.

The Intergenic Region Internal Ribosome Entry Sites (IGR IRESs) of Discistroviridae promote protein synthesis without initiation factors, with IRES translocation by elongation factor 2 (eEF2) being the first factor-catalysed reaction. Here, we developed a system that allows for the observation of intersubunit conformation of eukaryotic ribosomes at the single-molecule level by labeling rRNA. We used it to follow translation initiation and subsequent translocation of the cricket paralysis virus IRES (CrPV IRES). We observed that pre-translocation 80S-IRES ribosomes spontaneously exchanged between non-rotated and semi-rotated conformations, but predominantly occupied a semi-rotated conformation. In the presence of eEF2, ribosomes underwent forward and reverse translocation. Both reactions were eEF2 concentration dependent, indicating that eEF2 promoted both forward and reverse translocation. The antifungal, sordarin, stabilizes eEF2 on the ribosome after GTP hydrolysis in an extended conformation. 80S-CrPV IRES-eEF2-sordarin complexes underwent multiple rounds of forward and reverse translocations per eEF2 binding event. In the presence of sordarin, neither GTP hydrolysis nor a phosphate release were required for IRES translocation. Together, these results suggest that in the presence of sordarin, eEF2 promotes the mid and late stages of CrPV IRES translocation by unlocking ribosomal movements, with mid and late stages of translocation being thermally driven.