IRES Elements Research Articles

Genetic modification of primary human B cells to model the process of B cell development in germinal centers

The main stages of maturation of antigen-specific B cells occur in the germinal centers of the lymph nodes. During the process of differentiation, a decision is made on which path the B cells will take to develop further. They will either turn into short-lived plasmablasts or memory B cells or plasma cells. The relationship between these processes is very important for the development of a productive humoral immune response. The goal of the work was to create a system that is capable of simulating ex vivo processes occurring in germinal centers. We used primary B cells from human peripheral blood as starting material. B lymphocytes were stimulated in vitro using feeder cells carrying CD40L molecules and recombinant IL-21. Upon IL-21/CD40L stimulation, B lymphocytes changed their morphology, surface phenotype, and functional activity. After active expansion for 10 days, further cell growth stopped, and after some time they died. To generate stably proliferating B cells, we used lentiviral transduction of IL-21/CD40L stimulated IgM+ B cells. For this purpose, lentivirus preparations were obtained that carried a cassette consisting of the BCL6 and BCL2L1 genes, separated by a sequence encoding the self-cutting peptide P2A, as well as a GFP reporter gene separated from the target genes by an IRES element. The cassette used ensured the synthesis of the Bcl-6 transcription factor and the Bcl-XL protein in target cells. The Bcl-6 repressor prevented B cells from undergoing terminal differentiation and becoming plasma cells, and the Bcl-XL protein had an anti-apoptotic effect. Transduced B cells proliferated for more than a month and maintained a plasmablast phenotype. Forty-two days after the start of stimulation, transduced B cells remained GFP-positive, coexpressed CD27 and CD38 antigens, carried surface CD20 and IgM, intracellular Bcl-6, Bcl-XL and IgM, retained IgM secretion, but remained negative for surface and intracellular IgG. The proven stimulation system will allow us to simulate key aspects of B cell development in germinal centers to study the formation of B cell memory, which will ultimately facilitate the development of effective vaccines.

CircTRIM1 encodes TRIM1-269aa to promote chemoresistance and metastasis of TNBC via enhancing CaM-dependent MARCKS translocation and PI3K/AKT/mTOR activation

Peptides and proteins encoded by noncanonical open reading frames (ORFs) of circRNAs have recently been recognized to play important roles in disease progression, but the biological functions and mechanisms of these peptides and proteins are largely unknown. Here, we identified a potential coding circular RNA, circTRIM1, that was upregulated in doxorubicin-resistant TNBC cells by intersecting transcriptome and translatome RNA-seq data, and its expression was correlated with clinicopathological characteristics and poor prognosis in patients with TNBC. CircTRIM1 possesses a functional IRES element along with an 810 nt ORF that can be translated into a novel endogenously expressed protein termed TRIM1-269aa. Functionally, we demonstrated that TRIM1-269aa, which is involved in the biological functions of circTRIM1, promoted chemoresistance and metastasis in TNBC cells both in vitro and in vivo. In addition, we found that TRIM1-269aa can be packaged into exosomes and transmitted between TNBC cells. Mechanistically, TRIM1-269aa enhanced the interaction between MARCKS and calmodulin, thus promoting the calmodulin-dependent translocation of MARCKS, which further initiated the activation of the PI3K/AKT/mTOR pathway. Overall, circTRIM1, which encodes TRIM1-269aa, promoted TNBC chemoresistance and metastasis by enhancing MARCKS translocation and PI3K/AKT/mTOR activation. Our investigation has yielded novel insights into the roles of protein-coding circRNAs and supported circTRIM1/TRIM1-269aa as a novel promising prognostic and therapeutic target for patients with TNBC.

Decoding of translation-regulating entities reveals heterogeneous translation deficiency patterns in cellular senescence.

Cellular senescence constitutes a generally irreversible proliferation barrier, accompanied by macromolecular damage and metabolic rewiring. Several senescence types have been identified based on the initiating stimulus, such as replicative (RS), stress-induced (SIS) and oncogene-induced senescence (OIS). These senescence subtypes are heterogeneous and often develop subset-specific phenotypes. Reduced protein synthesis is considered a senescence hallmark, but whether this trait pertains to various senescence subtypes and if distinct molecular mechanisms are involved remain largely unknown. Here, we analyze large published or experimentally produced RNA-seq and Ribo-seq datasets to determine whether major translation-regulating entities such as ribosome stalling, the presence of uORFs/dORFs and IRES elements may differentially contribute to translation deficiency in senescence subsets. We show that translation-regulating mechanisms may not be directly relevant to RS, however uORFs are significantly enriched in SIS. Interestingly, ribosome stalling, uORF/dORF patterns and IRES elements comprise predominant mechanisms upon OIS, strongly correlating with Notch pathway activation. Our study provides for the first time evidence that major translation dysregulation mechanisms/patterns occur during cellular senescence, but at different rates depending on the stimulus type. The degree at which those mechanisms accumulate directly correlates with translation deficiency levels. Our thorough analysis contributes to elucidating crucial and so far unknown differences in the translation machinery between senescence subsets.

HIV-1 Tat impairs the establishment and maintenance of latency in a quiescent T cell line model of HIV infection

Abstract Human Immunodeficiency Virus (HIV) persists as a transcriptionally silent, latent provirus despite optimal therapy. Latently infected cells avoid immune detection and viral cytopathic effects becoming a long-lived reservoir of HIV. To elucidate factors responsible for the establishment of latency, our lab developed two novel HIV-1 reporter constructs (89.6 VT1 and 454 VT2). Studies using these reporters demonstrated that the multiplicity of infection (MOI) (the ratio of viral particles to cells) strongly correlated with the proportion of active versus latent infection in a “quiescent” T cell line. This observation suggested the hypothesis that at low viral inoculum, the availability of a viral factor, such as the HIV transactivator of transcription (Tat), was limiting and affected the likelihood that active versus latent infection was established. To test this, we (1) examined relative tat levels in reporter viruses that established different proportions of latent versus active infection (2) measured active versus latent infection in a cell line that constitutively overexpressed tat and (3) modified the dual reporter 89.6 VT1 to overexpress tat after an IRES element. In every case, higher tat levels correlated with increased active infection and a reduction in dependency of active infection on the amount of inoculum. Ongoing studies will test the extent to which Tat levels vary in native HIV proviral genomes. These studies provide insight into the molecular mechanisms that govern latency establishment at high versus low viral infection conditions and may shed light on new approaches to eradicate latently infected cells. Supported by grants from NIH/ NIAID (R01 AI148084 S1)

44P In situ CAR therapy using oRNA™ lipid nanoparticles regresses tumors in mice

LNP-mediated delivery of long coding RNA has been clinically validated for vaccines and gene editing. We have been developing a novel, synthetic, circular coding RNA platform (oRNA technology) which exhibits significant improvements in production, expression and formulation compared to mRNAs. Lacking the cap structure of mRNA, our oRNA technology uses a proprietary sequence-based IRES element to initiate protein translation in target cells. At the same time, ex vivo generated chimeric antigen receptor (CAR) T cell therapies have had tremendous success in treating hematologic malignancies, yet manufacturing, safety and efficacy challenges remain.

The introduction of mutations in the wild type coxsackievirus B3 (CVB3) IRES RNA leads to different levels of in vitro reduced replicative and translation efficiencies.

Coxsackievirus B3 (CVB3) is a principal causative agent of viral myocarditis, meningitis and pancreatitis. There is no vaccine available for clinical use. It has been demonstrated that the primary molecular determinant of virulence phenotype is located in the 5’ UTR of the viral genome. Translation initiation of CVB3 RNA is directed by the IRES element situated in the 5’UTR. In the present study, we analyse the effects of single point mutations introduced in different positions in the domain V of the IRES RNA of CVB3 wild type. We characterize in vitro virus replicative capacitiy and translation efficiency and we test in vivo virulence of different CVB3 mutants produced by the introduction of different mutations in the domain V of IRES by site-directed mutagenesis to abolish its structure. Our results demonstrate that all RNA mutants display different levels of decreased replication and translation initiation efficiency in vitro. The translation defect was correlated with significant reduced viral titer of mutant particles in comparison with the wild type. When inoculated in mice, mutant viruses were checked for inflammation and necrosis.In vitro and in vivo Findings strongly suggest that the most attenuated mutant strain could be considered a candidate for live-attenuated CVB3 vaccine.

Opposing effects of metformin mediated mTORC1 inhibition on IRES possessing anti-apoptotic proteins in breast cancer cell lines

Anti-diabetic drug, metformin, has attracted attentions as an anti-cancer agent due to the suppression of mTORC1 as a master switch of cell growth in various cancers. Upon mTORC1 inhibition, translation of proteins possessing IRES elements in their mRNAs still occurs in order to maintain proper cell function. Thus, inhibitor of apoptosis proteins are expected to be upregulated by mTORC1 inhibition due to the possession of IRES elements. Surprisingly, however, inhibition of mTORC1 with metformin in breast cancer cell lines of MDA-MB-231 and MCF-7, caused the downregulation of IRES-element possessing proteins of cIAP1, XIAP and Bcl-2, 24 h post-treatment. Interestingly, a shorter treatment time of 8 h, however, was accompanied by increased expression of these proteins. Importantly, inclusion of the proteasome inhibitor, Bortezomib, caused the upregulation of the tested anti-apoptotic proteins in both cell lines. These observations suggest that mTORC1 inhibition has a bifold effect; first upregulation of IRES-dependent survival proteins to prevent untimely cell death followed by an opposite effect, which is the enhanced proteosomal degradation of these protein in order to maintain a balanced response.

A Single Mutation in the Cryptic AUG (cAUG) Affects In Vitro Translation and Replication Efficiencies and In Vivo Virulence of Coxsackievirus B3 (CVB3).

The 5'UTR of the genomic RNA of CVB3, unusually long and rich on highly structured secondary structure, contains a conserved cis acting RNA element named the cryptic AUG (cAUG), where the cellular 48S complex is formed. In this study, we investigate the role of this cAUG in CVB3 translation, replication, and virulence. Mutant viral sub-genomic replicon RNA was constructed by site-directed mutagenesis. We characterize in vitro translation and replication efficiencies and in vivo virulence of a cAUG mutant in comparison with wild-type strain. UV-cross-linking assay and Real-Time PCR were used, respectively, to detect binding host proteins and to quantify viral production. Secondary structures of domain containing the cAUG site were studied and compared. The results suggest that introduced mutation in the CVB3 5'UTR affects in vitro and ex vivo viral translation which cannot be rescued by compensatory mutations. A reduced interaction of the La and PCBP2 translation initiation factors with cAUG residue of mutant was revealed. Decreasing production of viral mutant RNA was also demonstrated. Furthermore, secondary structure prediction reveals changes in the ribosome binding sites of the cAUG moiety of mutant sense strand RNA and no alterations in the structure of wild type, suggesting that cAUG mutation specifically affects the secondary structure of the sense RNA strand. Taken together, AUG integrity influences the efficiency of ribosome recruitment through IRES element and the capacity of replication.

SFPQ promotes RAS-mutant cancer cell growth by modulating 5'-UTR mediated translational control of CK1α.

Oncogenic mutations in the RAS family of small GTPases are commonly found in human cancers and they promote tumorigenesis by altering gene expression networks. We previously demonstrated that Casein Kinase 1α (CK1α), a member of the CK1 family of serine/threonine kinases, is post-transcriptionally upregulated by oncogenic RAS signaling. Here, we report that the CK1α mRNA contains an exceptionally long 5′-untranslated region (UTR) harbouring several translational control elements, implicating its involvement in translational regulation. We demonstrate that the CK1α 5′-UTR functions as an IRES element in HCT-116 colon cancer cells to promote cap-independent translation. Using tobramycin-affinity RNA-pulldown assays coupled with identification via mass spectrometry, we identified several CK1α 5′-UTR-binding proteins, including SFPQ. We show that RNA interference targeting SFPQ reduced CK1α protein abundance and partially blocked RAS-mutant colon cancer cell growth. Importantly, transcript and protein levels of SFPQ and other CK1α 5′-UTR-associated RNA-binding proteins (RBPs) are found to be elevated in early stages of RAS-mutant cancers, including colorectal and lung adenocarcinoma. Taken together, our study uncovers a previously unappreciated role of RBPs in promoting RAS-mutant cancer cell growth and their potential to serve as promising biomarkers as well as tractable therapeutic targets in cancers driven by oncogenic RAS.

Evaluation of a Novel DNA Vaccine Double Encoding Somatostatin and Cortistatin for Promoting the Growth of Mice.

Simple SummaryGrowth trait is one of the most important economic traits for meat animals. Somatostatin DNA vaccine has been proven to enhance the growth rate of animals. However, the growth-promoting effect is not ideal. This study aimed to evaluate the immune effects of a novel eukaryotic dual expression vaccine known as pIRES-S/CST14-S/2SS in mice. Firstly, we demonstrated pIRES-S/CST14-S/2SS could functionally express in GH3 pituitary cells by regulating the growth hormone (GH) and prolactin (PRL) productions. Secondly, we observed that all concentrations of pIRES-S/CST14-S/2SS vaccine could evoke anti-somatostatin (SS) antibodies, leading to a higher level of GH concentration. Notably, pIRES-S/CST14-S/2SS (10 μg/100 μL) immunized mice obtained maximum body weight gain in a booster vaccination period. Our results are helpful for better understanding of the relationship between SS and CST, and pIRES-S/CST14-S/2SS vaccine may be used as a promising alternative for developing growth-promoting vaccine.Animal growth traits are directly linked with the economics of livestock species. A somatostatin DNA vaccine has been developed to improve the growth of animals. However, the growth-promoting effect is still unsatisfying. The current study aimed to evaluate the effect of a novel eukaryotic dual expression vaccine known as pIRES-S/CST14-S/2SS, which encodes the genes obtained by fusing somatostatin (SS) and cortistatin (CST) into hepatitis B surface antigen (HBsAg). After transfection into GH3 cells with pIRES-S/CST14-S/2SS, green fluorescence signals were observed by fluorescence microscopy, suggesting the effective expression of CST and SS in GH3 cells using the IRES elements. Subsequently, both GH and PRL levels were found to be significantly lower in pIRES-S/CST14-S/2SS-treated cells as compared to the control group (p < 0.05). Furthermore, the antibody level, hormone secretion, and weight gain in the mice injected with novel recombinant plasmids were also evaluated. The anti-SS antibodies were detectable in all vaccine treated groups, resulting in significantly higher levels of GH secretion (p < 0.05). It is worth mentioning that pIRES-S/CST14-S/2SS (10 μg/100 μL) vaccinated mice exhibited a higher body weight gain in the second immunization period. This study increases the understanding of the relationship between somatostatin and cortistatin, and may help to develop an effective growth-promoting DNA vaccine in animals.

Suppression of the Inhibitory Effect of circ_0036176-Translated Myo9a-208 on Cardiac Fibroblast Proliferation by miR-218-5p.

Increasing evidence has shown that circular RNAs (circRNAs) participate in the process of cardiac remodeling. CircRNA circ_0036176 originating from the back-splicing of exon 2 to exon4 of myosin IXA (Myo9a) gene was shown to be increased in the myocardium of patients with heart failure (HF) and riched in exosomes from human AC16 cardiomyocytes with overexpression of circ_0036176. Proliferation activity was inhibited in mCFs subjected to exosomal circ_0036176 treatment and in mCFs with overexpression of circ_0036176. Interestingly, circ_0036176 contains an IRES element and an ORF of 627 nt encoding a 208-amino acid protein (termed as Myo9a-208). Myo9a-208 was shown to mediate the inhibitory effect of circ_0036176 on CFs proliferation, and miR-218-5p could inhibit Myo9a-208 expression by binding to circ_0036176, resulting in abolishing the effect of circ_0036176 on inactivating cyclin/Rb signal and suppressing CFs proliferation. Our findings suggest that circ_0036176 inhibits mCFs proliferation by translating Myo9a-208 protein to suppress cyclin/Rb pathway.

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. In the original study, we tested this hypothesis, by placing 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:Here 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 by employing RT-qPCR that in the IRES vectors, the Fluc activity levels measured by the luciferase assay are an accurate proxy of 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 readthrough permissive sequences and of IRES elements, though these require a separate investigation.

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 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. In the original study, we tested this hypothesis, by placing 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: Here 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 by employing RT-qPCR that in the IRES vectors, the Fluc activity levels measured by the luciferase assay are an accurate proxy of 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 readthrough permissive sequences and of IRES elements, though these require a separate investigation.

Nucleolin Promotes IRES-Driven Translation of Foot-and-Mouth Disease Virus by Supporting the Assembly of Translation Initiation Complexes.

Nucleolin (NCL), a stress-responsive RNA-binding protein, has been implicated in the translation of internal ribosome entry site (IRES)-containing mRNAs, which encode proteins involved in cell proliferation, carcinogenesis, and viral infection (type I IRESs). However, the details of the mechanisms by which NCL participates in IRES-driven translation have not hitherto been described. Here, we identified NCL as a protein that interacts with the IRES of foot-and-mouth disease virus (FMDV), which is a type II IRES. We also mapped the interactive regions within FMDV IRES and NCL in vitro. We found that NCL serves as a substantial regulator of FMDV IRES-driven translation but not of bulk cellular or vesicular stomatitis virus cap-dependent translation. NCL also modulates the translation of and infection by Seneca Valley virus (type III-like IRES) and classical swine fever virus (type III IRES), which suggests that its function is conserved in unrelated IRES-containing viruses. We also show that NCL affects viral replication by directly regulating the production of viral proteins and indirectly regulating FMDV RNA synthesis. Importantly, we observed that the cytoplasmic relocalization of NCL during FMDV infection is a substantial step for viral IRES-driven translation and that NCL specifically promotes the initiation phase of the translation process by recruiting translation initiation complexes to viral IRES. Finally, the functional importance of NCL in FMDV pathogenicity was confirmed in vivo. Taken together, our findings demonstrate a specific function for NCL in selective mRNA translation and identify a target for the development of a broad-spectrum class of antiviral interventions. IMPORTANCE FMDV usurps the cellular translation machinery to initiate viral protein synthesis via a mechanism driven by IRES elements. It allows the virus to shut down bulk cellular translation, while providing an advantage for its own gene expression. With limited coding capacity in its own genome, FMDV has evolved a mechanism to hijack host proteins to promote the recruitment of the host translation machinery, a process that is still not well understood. Here, we identified nucleolin (NCL) as a positive regulator of the IRES-driven translation of FMDV. Our study supports a model in which NCL relocalizes from the nucleus to the cytoplasm during the course of FMDV infection, where the cytoplasmic NCL promotes FMDV IRES-driven translation by bridging the translation initiation complexes with viral IRES. Our study demonstrates a previously uncharacterized role of NCL in the translation initiation of IRES-containing viruses, with important implications for the development of broad antiviral interventions.

Abstract IA13: An mTOR/eIF4E-independent translation mechanism promotes breast cancer metastasis

Abstract While translation initiation of most mammalian mRNAs is mediated by a 5' cap structure that binds eIF4E, inactivation/silencing of eIF4E, or quantitative sequestration of eIF4E by 4E-BPs through mTORC1 inhibition, does not impair translation of many capped mRNAs. This suggests an alternate mechanism may exist for cap-dependent but eIF4E/mTORC1-independent translation. DAP5 (NAT1, eIF4G2, p97) is an eIF4G family member that lacks the N-terminal binding domains for eIF4E and PABP. DAP5 was shown to mediate translation initiation through internal ribosome entry sites (IRES elements). We recently showed that DAP5 utilizes the cap binding activity of eIF3d to facilitate cap-dependent translation of a large number of capped mRNAs, making it a previous unknown major mechanism of cap-dependent but eIF4E/mTORC1-independent mRNA translation. Genome-wide transcriptomic and translatomic analyses indicate that DAP5 is required for translation of many transcription factor and cell receptor capped mRNAs and their mRNA targets involved in cell survival, motility, DNA repair, oncogenesis, translation initiation, and cancer cell metastasis. Up to 20% of cellular mRNAs utilize DAP5-eIF3d for cap-dependent mRNA translation. Mass spectrometry and in vivo cross-linking studies found that eIF3d is a direct binding partner of DAP5 but does not bind strongly to eIF4GI or II family members that bind eIF4E. In vitro translation and ribosome complex studies demonstrate that DAP5 and eIF3d are both essential for eIF4E and mTORC1-independent capped-mRNA translation. Given the prevalence of oncogenic and survival mRNAs directed in translation by DAP5-eIF3d, we analyzed the NCI human tissue genome cancer atlas (TCGA) and found that high expression of DAP5 mRNA is strongly associated with estrogen receptor-negative (ER-) breast cancer metastasis and poor survival. We therefore investigated the role of DAP5 in preclinical mouse models of highly metastatic ER- breast cancer. Reduction of DAP5 expression by ~70% almost eliminated metastasis to the lung and significantly increased survival with no impact on primary tumor growth. These and other studies indicate that the DAP5-eIF3d complex is a significant alternate mechanism for cap-dependent, eIF4E/mTORC1-independent mRNA translation despite lacking eIF4E and PABP binding, that DAP5 directs translation of many capped mRNAs that are involved in cell stress and survival pathways, is essential for promoting breast cancer metastasis, and that most mRNAs translated by DAP5 do not utilize an IRES. Citation Format: Columba de la Parra, Amanda Ernlund, Amandine Alard, Kelly Ruggles, Beatrix Ueberheide, Olga Katsara, Robert J. Schneider. An mTOR/eIF4E-independent translation mechanism promotes breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA13.

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 (AMD1 tail). 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 governed by EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Methods: Here we employ dual luciferase assay and western blotting to explore the effects of AMD1 tail and control sequences on reporter 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. The decreased reporter activity that appears to be induced by the readthrough sequence occurs only in reporters containing EMCV IRES. Monocistronic reporters with the same readthrough context sequence exhibit only a modest reduction in reporter activity. Furthermore, in monocistronic vectors, the disproportionate reduction of reporter levels greatly increased when AMD1 tail was translated as a result of readthrough. Such readthrough-mediated reduction was not observed when AMD1 tail was substituted with unrelated sequences in agreement with our original hypothesis. 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 readthrough permissive sequences and of IRES elements, though these require a separate investigation.

Evaluation of different IRES-mediated tricistronic plasmid designs for expression of an anti-PCSK9 biosimilar monoclonal antibody in CHO cells.

To compare different approaches for the expression of an anti-PCSK9 biosimilar monoclonal antibody (mAb) in CHO cells using IRES-mediated tricistronic plasmid vectors combining different signal peptides, IRES elements and selection markers. Transient transfection indicated a similar level of secreted mAb 48h post-transfection for all constructs. However, transfections carried out with circular plasmids showed a higher expression than with linearized plasmids. After two months under selection pressure, only part of the transfected pools recovered. The cultures co-transfected using two antibiotics as selection markers for double selection did not recover. Growth, metabolism and mAb production profiles of the only part of the transfected pools recovered resulting stable pools were compared and the stable pool transfected with circular L1-LC-IRES-H7-HC-IRES-NEO plasmid was chosen for further studies, due to higher cell growth and mAb production. Critical quality attributes of the protein A-purified mAb such as purity, homogeneity, binding affinity to PCSK9, and amino acid sequence were assessed confirming the success of the approach adopted in this study. The expression platform proposed showed to be efficient to produce a high-quality anti-PCSK9 mAb in stable CHO cell pools and provides benchmarks for fast production of different mAbs for characterization, formulation studies and pre-clinical investigation.

Heterogeneous Nuclear Ribonucleoprotein L Negatively Regulates Foot-and-Mouth Disease Virus Replication through Inhibition of Viral RNA Synthesis by Interacting with the Internal Ribosome Entry Site in the 5′ Untranslated Region

Upon infection, the highly structured 5' untranslated region (5' UTR) of picornavirus is involved in viral protein translation and RNA synthesis. As a critical element in the 5' UTR, the internal ribosome entry site (IRES) binds to various cellular proteins to function in the processes of picornavirus replication. Foot-and-mouth disease virus (FMDV) is an important member in the family Picornaviridae, and its 5' UTR contains a functional IRES element. In this study, the cellular heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as an IRES-binding protein for FMDV by biotinylated RNA pulldown assays, mass spectrometry (MS) analysis, and determination of hnRNP L-IRES interaction regions. Further, we found that hnRNP L inhibited the growth of FMDV through binding to the viral IRES and that the inhibitory effect of hnRNP L on FMDV growth was not due to FMDV IRES-mediated translation, but to influence on viral RNA synthesis. Finally, hnRNP L was demonstrated to coimmunoprecipitate with RNA-dependent RNA polymerase (3Dpol) in an FMDV RNA-dependent manner in the infected cells. Thus, our results suggest that hnRNP L, as a critical IRES-binding protein, negatively regulates FMDV replication by inhibiting viral RNA synthesis, possibly by remaining in the replication complex.IMPORTANCE Picornaviruses, as a large family of human and animal pathogens, cause a bewildering array of disease syndromes. Many host factors are implicated in the pathogenesis of these viruses, and some proteins interact with the viral IRES elements to affect function. Here, we report for the first time that cellular hnRNP L specifically interacts with the IRES of the picornavirus FMDV and negatively regulates FMDV replication through inhibiting viral RNA synthesis. Further, our results showed that hnRNP L coimmunoprecipitates with FMDV 3Dpol in a viral RNA-dependent manner, suggesting that it may remain in the replication complex to function. The data presented here would facilitate further understanding of virus-host interactions and the pathogenesis of picornavirus infections.

A conserved RNA structural motif for organizing topology within picornaviral internal ribosome entry sites

Picornaviral IRES elements are essential for initiating the cap-independent viral translation. However, three-dimensional structures of these elements remain elusive. Here, we report a 2.84-Å resolution crystal structure of hepatitis A virus IRES domain V (dV) in complex with a synthetic antibody fragment—a crystallization chaperone. The RNA adopts a three-way junction structure, topologically organized by an adenine-rich stem-loop motif. Despite no obvious sequence homology, the dV architecture shows a striking similarity to a circularly permuted form of encephalomyocarditis virus J-K domain, suggesting a conserved strategy for organizing the domain architecture. Recurrence of the motif led us to use homology modeling tools to compute a 3-dimensional structure of the corresponding domain of foot-and-mouth disease virus, revealing an analogous domain organizing motif. The topological conservation observed among these IRESs and other viral domains implicates a structured three-way junction as an architectural scaffold to pre-organize helical domains for recruiting the translation initiation machinery.