Cap-independent Translation Element Research Articles

Host-like RNA Elements Regulate Virus Translation.

Viruses are obligate, intracellular parasites that co-opt host cell machineries for propagation. Critical among these machineries are those that translate RNA into protein and their mechanisms of control. Most regulatory mechanisms effectuate their activity by targeting sequence or structural features at the RNA termini, i.e., at the 5' or 3' ends, including the untranslated regions (UTRs). Translation of most eukaryotic mRNAs is initiated by 5' cap-dependent scanning. In contrast, many viruses initiate translation at internal RNA regions at internal ribosome entry sites (IRESs). Eukaryotic mRNAs often contain upstream open reading frames (uORFs) that permit condition-dependent control of downstream major ORFs. To offset genome compression and increase coding capacity, some viruses take advantage of out-of-frame overlapping uORFs (oORFs). Lacking the essential machinery of protein synthesis, for example, ribosomes and other translation factors, all viruses utilize the host apparatus to generate virus protein. In addition, some viruses exhibit RNA elements that bind host regulatory factors that are not essential components of the translation machinery. SARS-CoV-2 is a paradigm example of a virus taking advantage of multiple features of eukaryotic host translation control: the virus mimics the established human GAIT regulatory element and co-opts four host aminoacyl tRNA synthetases to form a stimulatory binding complex. Utilizing discontinuous transcription, the elements are present and identical in all SARS-CoV-2 subgenomic RNAs (and the genomic RNA). Thus, the virus exhibits a post-transcriptional regulon that improves upon analogous eukaryotic regulons, in which a family of functionally related mRNA targets contain elements that are structurally similar but lacking sequence identity. This "thrifty" virus strategy can be exploited against the virus since targeting the element can suppress the expression of all subgenomic RNAs as well as the genomic RNA. Other 3' end viral elements include 3'-cap-independent translation elements (3'-CITEs) and 3'-tRNA-like structures. Elucidation of virus translation control elements, their binding proteins, and their mechanisms can lead to novel therapeutic approaches to reduce virus replication and pathogenicity.

Crystal structure of a cap-independent translation enhancer RNA.

In eukaryotic messengerRNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV2) RNA in complex with our structural chaperone, Fab BL3-6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV2 tertiary architecture is conserved among these homologs.

Insights into the secondary and tertiary structure of the Bovine Viral Diarrhea Virus Internal Ribosome Entry Site

ABSTRACT The internal ribosome entry site (IRES) RNA of bovine viral diarrhoea virus (BVDV), an economically significant Pestivirus, is required for the cap-independent translation of viral genomic RNA. Thus, it is essential for viral replication and pathogenesis. We applied a combination of high-throughput biochemical RNA structure probing (SHAPE-MaP) and in silico modelling approaches to gain insight into the secondary and tertiary structures of BVDV IRES RNA. Our study demonstrated that BVDV IRES RNA in solution forms a modular architecture composed of three distinct structural domains (I–III). Two regions within domain III are represented in tertiary interactions to form an H-type pseudoknot. Computational modelling of the pseudoknot motif provided a fine-grained picture of the tertiary structure and local arrangement of helices in the BVDV IRES. Furthermore, comparative genomics and consensus structure predictions revealed that the pseudoknot is evolutionarily conserved among many Pestivirus species. These studies provide detailed insight into the structural arrangement of BVDV IRES RNA H-type pseudoknot and encompassing motifs that likely contribute to the optimal functionality of viral cap-independent translation element.

Multiplexed Analysis of Human uORF Regulatory Functions During the ISR Using PoLib-Seq.

Protein synthesis is a highly regulated essential process. As such, it is subjected to substantial regulation in response to stress. One hallmark of the Integrated Stress Response (ISR) is the immediate shutdown of most translation through phosphorylation of the alpha subunit of translation initiation factor eIF2 and activation of eIF4E binding proteins. While these posttranslational modifications largely inhibit cap-dependent translation, many mRNA resist this inhibition by alternative translation mechanisms involving cis-regulatory sequences and structures in 5' transcript leaders, including upstream Open Reading Frames (uORFs), Internal Ribosome Entry Sites (IRESes), and Cap-Independent Translation Elements (CITEs). Studies of uORF and IRES activity are often performed on a gene-by-gene basis; however, high-throughput methods have recently emerged. Here, we describe a protocol for Polysome Library Sequencing (PoLib-Seq; Fig. 1), a multiplexed assay of reporter gene translation that can be used during the ISR. A designer library of reporter RNAs are transfected into tissue-culture cells, and their translation is assayed via sucrose gradient fractionation followed by high-throughput sequencing. As an example, we include PoLib-seq results simultaneously assaying translation of wildtype and uORF mutant human ATF4 reporter RNAs, recapitulating the known function of uORF1 in resisting translational inhibition during the ISR.

Binding of eukaryotic initiation factor 3 (eIF3) to Barley Yellow Dwarf Virus (BYDV) untranslated regions (UTRs)

Viruses remain a major threat to human health and the global food sources. Viruses are obligate parasites that use their host's cellular machinery to translate their proteins, usually employing unique non-canonical translation mechanisms to outcompete the host cell mRNA for this machinery. BYDV is a positive-strand RNA virus that lacks the canonical 5ʹ 7-methylguanosine cap and a poly-A tail in its mRNA, instead utilizing a CITE (cap-independent translation element) in the 3ʹ UTR, termed as the 3ʹ BTE (BYDV like CITE). The BYDV 3ʹ BTE is 105-nt long and has a cruciform secondary structure with three stem loops SL-I, SL-II, SL-III and a base stem (SL-IV). BTE has been shown to use the host cell's eukaryotic translation initiation factor (eIF) 4G, eIF4A, eIF4B and ATP (an active helicase complex) to recruit the 40S ribosomal subunit. This 3ʹ BTE-recruited translation initiation complex must transfer to the 5ʹ end of the mRNA for scanning, AUG detection, and translation elongation to proceed. The 5ʹ UTR of BYDV is also structured, consisting of four stem loops, (SL-A, B, C and D), and the 3ʹ to 5ʹ transfer process is aided by a relatively weak, long-distance, ‘kissing-loop’, base pairing interaction between stem-loops in the 5ʹ and 3ʹ ends of BYDV. Recently, we showed that eIF3 plays a crucial role in this transfer by simultaneously binding to both the BYDV 5ʹ and 3ʹ UTRs. These binding interactions were found to be helicase dependent, and the UTRs did not compete for eIF3 binding, suggesting the presence of a transient complex. eIF3 has been shown to interact with both the 5ʹ IRES (Internal ribosome entry site) and 3ʹ UTR of Hepatitis C Virus (HCV) simultaneously. In HCV-like IRESs, the eIF3 and the IRES binding site on the 40S subunit has been shown to overlap, requiring structural rearrangements and dislocation of eIF3 for IRES to bind. In BYDV, we expect similar rearrangements in the UTRs for eIF3 to successfully transfer the 40S complex from the 3ʹ to the 5ʹ end. To understand this macromolecular complex consisting of eIF3, the helicase complex, and the BYDV UTRs, we used a fusion RNA oligo comprising the BYDV 5ʹ UTR and 3ʹ BTE linked by a 12-base RNA linker and performed fluorescence anisotropy-based binding studies to eIF3. Interestingly, the helicase complex was not required for eIF3 binding to the Fusion oligo. SHAPE footprinting analyses performed on the fusion oligo showed that the presence of all helicase factors shifts the eIF3 binding site on the RNA closer to the kissing loop interaction between the UTRs. These data suggest that the helicase complex is not required for binding if the UTRs are in close proximity, but it does aid in rearranging the UTR RNAs to accommodate optimum eIF3 binding for eventual transfer of the 40S complex from the 3ʹ to the 5ʹ end of BYDV.

The RNA of Maize Chlorotic Mottle Virus, an Obligatory Component of Maize Lethal Necrosis Disease, Is Translated via a Variant Panicum Mosaic Virus-Like Cap-Independent Translation Element.

Maize chlorotic mottle virus (MCMV) combines with a potyvirus in maize lethal necrosis disease (MLND), a serious emerging disease worldwide. To inform resistance strategies, we characterized the translation initiation mechanism of MCMV. We report that MCMV RNA contains a cap-independent translation element (CITE) in its 3' untranslated region (UTR). The MCMV 3' CITE (MTE) was mapped to nucleotides 4164 to 4333 in the genomic RNA. 2'-Hydroxyl acylation analyzed by primer extension (SHAPE) probing revealed that the MTE is a distinct variant of the panicum mosaic virus-like 3' CITE (PTE). Like the PTE, electrophoretic mobility shift assays (EMSAs) indicated that eukaryotic translation initiation factor 4E (eIF4E) binds the MTE despite the absence of an m7GpppN cap structure, which is normally required for eIF4E to bind RNA. Using a luciferase reporter system, mutagenesis to disrupt and restore base pairing revealed that the MTE interacts with the 5' UTRs of both genomic RNA and subgenomic RNA1 via long-distance kissing stem-loop interaction to facilitate translation. The MTE stimulates a relatively low level of translation and has a weak, if any, pseudoknot, which is present in the most active PTEs, mainly because the MTE lacks the pyrimidine-rich tract that base pairs to a G-rich bulge to form the pseudoknot. However, most mutations designed to form a pseudoknot decreased translation activity. Mutations in the viral genome that reduced or restored translation prevented and restored virus replication, respectively, in maize protoplasts and in plants. In summary, the MTE differs from the canonical PTE but falls into a structurally related class of 3' CITEs.IMPORTANCE In the past decade, maize lethal necrosis disease has caused massive crop losses in East Africa. It has also emerged in China and parts of South America. Maize chlorotic mottle virus (MCMV) infection is required for this disease. While some tolerant maize lines have been identified, there are no known resistance genes that confer immunity to MCMV. In order to improve resistance strategies against MCMV, we focused on how the MCMV genome is translated, the first step of gene expression by all positive-strand RNA viruses. We identified a structure (cap-independent translation element) in the 3' untranslated region of the viral RNA genome that allows the virus to usurp a host translation initiation factor, eIF4E, in a way that differs from host mRNA interactions with the translational machinery. This difference indicates eIF4E may be a soft target for engineering of-or breeding for-resistance to MCMV.

Eukaryotic initiation factor (eIF) 3 mediates Barley Yellow Dwarf Viral mRNA 3'-5' UTR interactions and 40S ribosomal subunit binding to facilitate cap-independent translation.

Barley Yellow Dwarf Virus (BYDV) is a positive strand RNA virus that lacks the canonical 5′ 7-methylguanosine cap and a 3′ poly-A tail. Instead, BYDV utilizes a cruciform cap independent translation element (CITE) in its 3′UTR RNA (BYDV-like CITE or BTE) that binds eukaryotic translation initiation factor (eIF) 4F and recruits 40S ribosomal subunits in the presence of active helicase factors (eIF4A, eIF4B, eIF4F and ATP). A long-range, 5-nucleotide, base-pairing kissing loop interaction between the 3′BTE and a 5′UTR stem-loop is necessary for translation to initiate. The 40S–eIF complex does not bind to the BYDV 5′UTR, suggesting the involvement of additional factors. We identified eIF3 as a component of the 3′BTE recruited complex using affinity-tagged 3′BTE RNA pull-down assays. Fluorescence anisotropy binding and gel shift assays showed that the 3′BTE and 5′UTR RNAs can simultaneously and non-competitively bind eIF3 in the presence of active helicase factors forming a single, macromolecular complex. Further, quantitative studies showed eIF3 increased recruitment of the 40S subunit by more than 25-fold. We propose a new role for eIF3, where eIF3 bridges BYDV’s UTRs, stabilizes the long-range 5′-3′ interaction, and facilitates recruitment of the 40S–eIF complex to the 5′UTR, leading to translation initiation.

The 3' Untranslated Region of a Plant Viral RNA Directs Efficient Cap-Independent Translation in Plant and Mammalian Systems.

Many plant viral RNA genomes lack a 5′ cap, and instead are translated via a cap-independent translation element (CITE) in the 3′ untranslated region (UTR). The panicum mosaic virus-like CITE (PTE), found in many plant viral RNAs, binds and requires the cap-binding translation initiation factor eIF4E to facilitate translation. eIF4E is structurally conserved between plants and animals, so we tested cap-independent translation efficiency of PTEs of nine plant viruses in plant and mammalian systems. The PTE from thin paspalum asymptomatic virus (TPAV) facilitated efficient cap-independent translation in wheat germ extract, rabbit reticulocyte lysate, HeLa cell lysate, and in oat and mammalian (BHK) cells. Human eIF4E bound the TPAV PTE but not a PTE that did not stimulate cap-independent translation in mammalian extracts or cells. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) footprinting revealed that both human and wheat eIF4E protected the conserved guanosine (G)-rich domain in the TPAV PTE pseudoknot. The central G plays a key role, as it was found to be required for translation and protection from SHAPE modification by eIF4E. These results provide insight on how plant viruses gain access to the host’s translational machinery, an essential step in infection, and raise the possibility that similar PTE-like mechanisms may exist in mRNAs of mammals or their viruses.

Role of eukaryotic initiation factor 3 in the non‐canonical mechanism of Barley Yellow Dwarf Virus translation initiation

Plant viruses are a world‐wide threat both economically and to the food supply. Many of these are positive‐strand RNA viruses that have developed a myriad of non‐canonical translation initiation mechanisms. These unique mechanisms circumvent cap‐dependent translational controls and enable the viruses to outcompete host cell mRNA for the translation machinery. Many RNA viruses lack the canonical 5′ 7‐methylguanosine cap in their mRNAs, thereby allowing them to bypass canonical translation initiation. Instead, these mRNAs utilize structures in the 3′ untranslated region (UTR) of their mRNA known as a 3′ CITE (cap‐independent translation element) to drive non‐canonical translation initiation. Recently, we have shown that a 3′ CITE in Barley Yellow Dwarf Virus (BYDV), termed the 3′ BTE (BYDV 3′ CITE), can bind eukaryotic translation initiation factor (eIF) 4G, eIF4A, and ATP‐bound eIF4B in order to recruit the 40S ribosomal subunit and form a pre‐initiation complex (PIC) at the 3′ BTE. These eIFs cannot directly recruit the 40S subunit to the 5′ UTR of BYDV, but must instead transfer the 40S subunits to the 5′ end of the mRNA scanning, AUG detection, and translation elongation to proceed. This transfer process is aided by a long‐distance, ‘kissing‐loop’, base pairing interaction between stem‐loops in the 5′ and 3′ ends of BYDV. This kissing loop interaction is relatively weak, leading to the hypothesis that additional factors are required to create a stable 5′–3′ interaction capable of efficiently transferring the PIC. Data from pulldown experiments have shown that, in addition to eIF4G, almost every subunit of eIF3 is bound to the BTE. Single‐molecule fluorescence co‐localization experiments have shown that eIF3 specifically and significantly enhanced binding of 40S subunits to the BTE. Moreover, fluorescence anisotropy‐based binding studies have shown that eIF3 binds directly to both the BTE and the 5′ UTR of BYDV, that the BTE and 5′ UTR do not compete for binding to eIF3, and that the presence of the 40S subunit increases the affinity with which eIF3 binds to the BTE and 5′ UTR. Collectively, these data suggest that eIF3, the 40S subunit, the 3′ BTE, and the 5′ UTR are components of a single macromolecular complex. Based on this interpretation of our data, we propose that eIF3 bridges the 3′ and 5′ ends of BYDV and aids the kissing loop interaction in transferring the PIC from the 3′ to the 5′ end of BYDV.Support or Funding InformationGrant Support: NSF MCB‐1513737 (to DJG), NSF‐IGERT Fellowship 0965983 (to PP), and NIH GM 084288 (to RLG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural alteration of a BYDV-like translation element (BTE) that attenuates p35 expression in three mild Tobacco bushy top virus isolates

To identify the molecular effects of Tobacco bushy top virus (TBTV) evolution on the degeneration of tobacco bushy top disease, three TBTV isolates with mild virulence were compared with wild-type TBTV to assess the translation of p35, which relies on a BYDV-like translation element (BTE) in a cap-independent manner. The in vitro expression of p35 in the mild isolates was only 20% to 40% of the expression observed in wt TBTV. Based on translation data from chimeric TBTV RNA, low-level p35 expression in the three mild isolates was associated with two regions: the 5′ terminal 500 nt region (RI) and the 3′ internal region (RV), which included the BTE. For the RV region, low level p35 expression was mainly associated with structural alterations of the BTE instead of specific sequence mutations within the BTE based on SHAPE structural probing and mutation analysis. Additionally, structural alteration of the TBTV BTE resulted from mutations outside of the BTE, implying structural complexity of the local region surrounding the BTE. This study is the first report on the structural alteration of the 3′ cap-independent translation element among different isolates of a given RNA virus, which is associated with variations in viral virulence.

Eukaryotic translation initiation factor 4G (eIF4G) coordinates interactions with eIF4A, eIF4B, and eIF4E in binding and translation of the barley yellow dwarf virus 3′ cap-independent translation element (BTE)

Barley yellow dwarf virus RNA, lacking a 5' cap and a 3' poly(A) tail, contains a cap-independent translation element (BTE) in the 3'-untranslated region that interacts with host translation initiation factor eIF4G. To determine how eIF4G recruits the mRNA, three eIF4G deletion mutants were constructed: (i) eIF4G601-1196, containing amino acids 601-1196, including the putative BTE-binding region, and binding domains for eIF4E, eIF4A, and eIF4B; (ii) eIF4G601-1488, which contains an additional C-terminal eIF4A-binding domain; and (iii) eIF4G742-1196, which lacks the eIF4E-binding site. eIF4G601-1196 binds BTE tightly and supports efficient translation. The helicase complex, consisting of eIF4A, eIF4B, and ATP, stimulated BTE binding with eIF4G601-1196 but not eIF4G601-1488, suggesting that the eIF4A binding domains may serve a regulatory role, with the C-terminal binding site having negative effects. eIF4E binding to eIF4G601-1196 induced a conformational change, significantly increasing the binding affinity to BTE. A comparison of the binding of eIF4G deletion mutants with BTEs containing mutations showed a general correlation between binding affinity and ability to facilitate translation. In summary, these results reveal a new role for the helicase complex in 3' cap-independent translation element-mediated translation and show that the functional core domain of eIF4G plus an adjacent probable RNA-binding domain mediate translation initiation.

Ligand-responsive upregulation of 3' CITE-mediated translation in a wheat germ cell-free expression system.

We have rationally constructed a novel regulation-type of artificial riboswitch that ligand-dose dependently upregulates translation initiation mediated by a 3' cap-independent translation element (3' CITE) with no major hybridization switches in a plant expression system (wheat germ extract).

Noncoding RNAs of Plant Viruses and Viroids: Sponges of Host Translation and RNA Interference Machinery.

Noncoding sequences in plant viral genomes are well-known to control viral replication and gene expression in cis. However, plant viral and viroid noncoding (nc)RNA sequences can also regulate gene expression acting in trans, often acting like 'sponges' that bind and sequester host cellular machinery to favor viral infection. Noncoding sequences of small subgenomic (sg)RNAs of Barley yellow dwarf virus (BYDV) and Red clover necrotic mosaic virus (RCNMV) contain a cap-independent translation element that binds translation initiation factor eIF4G. We provide new evidence that a sgRNA of BYDV can globally attenuate host translation, probably by sponging eIF4G. Subgenomic ncRNA of RCNMV is generated via 5' to 3' degradation by a host exonuclease. The similar noncoding subgenomic flavivirus (sf)RNA, inhibits the innate immune response, enhancing viral pathogenesis. Cauliflower mosaic virus transcribes massive amounts of a 600-nt ncRNA, which is processed into small RNAs that overwhelm the host's RNA interference (RNAi) system. Viroids use the host RNAi machinery to generate viroid-derived ncRNAs that inhibit expression of host defense genes by mimicking a microRNA. More examples of plant viral and viroid ncRNAs are likely to be discovered, revealing fascinating new weaponry in the host-virus arms race.

Identification of the functional binding domains of eukaryotic initiation factor eIF4G with 3’ Cap‐independent translation element of Barley Yellow Dwarf Virus (LB203)

Different from canonical translation initiation, some plant viral RNAs lack a 5’cap (an unusual 7‐methyl guanosine linked by a 5’to 5’ triphosphate). They utilize a cap independent translation element (CITE) to efficiently start translation. Barley yellow dwarf virus (BYDV) has a translation element (BTE) located in its 3’UTR mRNA. BTE binds with eukaryotic initiation factor eIF4G (one subunit of heterodimer of eIF4F) and recruits other initiation factors to assemble the ribosome preinitiation complex for synthesis of viral proteins. BYDV is one of the world‐wide economically most important viruses causing severe crop loss. How BTE mediate BYDV translation is still unclear. By measuring fluorescence anisotropy of labeled RNA, here we report 3’BTE binds with a truncated fragment of eIF4G, eIF4G601‐1196 (contained amino acid residue from 601 to 1196). The equilibrium dissociation constants (Kd) of this binding is 39.5±3.5 nM. Adding eIF4E, the cap binding protein, can increase the binding ability of eIF4G601‐1196 with 3’BTE. eIF4G601‐1196•4E complex binds with 3’BTE 5.8 folds tighter than eIF4G601‐1196 alone, with the equilibrium dissociation constant 6.78±1.49nM. Further circular dichroism spectra (CD) and thermodynamic studies will characterize the binding of the eIF4G with 3’ BTE. These results suggest eIF4E plays an indirect role in translation initiation, even for an uncapped virus.Grant Funding Source: NSF MCB 1157632

Understanding the role of pea enation mosaic virus mRNA 3’ untranslated region in translation initiation (566.5)

Most eukaryotic mRNAs use two key elements at opposite ends of the mRNA, the 5’ m7GpppN cap and the 3’ poly adenosine (A) tail, to recruit translation initiation machinery and circularize mRNA. Pea Enation Mosaic Virus (PEMV) mRNA lacks both a 5’ m7GpppN cap and a 3’ poly (A) tail. A novel type of cap independent translation elements (PTE) is found in the 3’UTR and is essential for translation initiation, the detailed mechanisms of initiation are not well known. Direct protein fluorescence quenching and labeled RNA fluorescence anisotropy were used to investigate the binding affinity of PTE fragment with two important eukaryotic initiation factors, eIF4E (Kd=~7.2nM) and eIF4F (Kd=~8.4nM). These binding affinities are as high as those for eIF4E and eIF4F binding with the cap analogue m7GTP and much higher than those for m7GpppG. With the fluorescence anisotropy assays, we also demonstrated that eIF4A, eIF4B and Poly A tail binding protein (PABP), which can form a stable complex with eIF4F in vivo, only slightly increased the binding affinity of eIF4F with the PTE RNA (Kd<5nM). These binding affinities suggest the PTE element can sequester eIFs from the host cell. The role of eIF/PTE binding in ribosome assembly is being investigated.Grant Funding Source: NSF MCB 1157632

Elongation factor 1A binds the 3’ translation element of the barley yellow dwarf virus (566.4)

The Barley Yellow Dwarf Virus (BYDV) is especially devastating due to its global widespread distribution and its infection of staple cereal crops. Our research aims to understand mechanisms of BYDV mRNA translation to gain insight into combating the disease and its dire economic implications. Previous studies have shown that BYDV initiates translation in a way that is unique to most eukaryotic messages. Almost all eukaryotic mRNA consist of a 5’ cap and initiates translation at the 5’ untranslated region (UTR). BYDV mRNA, however, consists of a cap‐independent translation element at the 3’ UTR (BTE). More interestingly, eukaryotic initiation factor 4F (eI4F) binds the 3’ end of BYDB mRNA, at the BTE, and translation occurs when the 3’ UTR interacts with the 5’ UTR. Pull‐down experiments using the BTE RNA as bait have revealed not only eIF4F binding but also a small array of other proteins not present when a nonfunctional BTE (BTEBF) or the 5’ UTR is used as bait. One prominent protein that appears to bind BTE but not the control BTEBF or 5’ UTR RNA is an unknown 50 kDa protein. Using Mass Spectrometry, we identify this protein to be elongation factor 1A (EF1A). EF1A has previously been shown to bind the 3’ UTR of Tobacco Etch Virus (TEV) mRNA and may have a role in in regulation and replication of the viral genes.Grant Funding Source: NSF MCB 1157632

Barley yellow dwarf virus (BYDV) utilizes a novel strategy for translation initiation.

Most commonly, gene expression in uncapped RNA viruses is regulated by either an internal ribosomal entry site (IRES) or cap independent translation element (CITE), which are located mostly in the 5′ untranslated region (UTR) of viral mRNA. BYDV, which lacks both a cap and poly(A) tail, has a CITE (called BTE) present in its 3′UTR that is essential for efficient translation initiation at the 5′ proximal AUG. The mechanism of BYDV translation initiation is not understood.Using fluorescence anisotropy and gel mobility shift assays we demonstrated that the 40S ribosome has moderate binding affinity to the 3′ UTR (Kd~600nM) and has extremely low affinity for any other parts of mRNA. The binding affinity to 3′UTR increases nearly six fold (Kd~100nM) in the presence of the eIF4F‐4B‐4A‐ATP helicase complex. In order to identify the structural elements that interact with initiation factors or ribosomes, the secondary structure of both 3′ and 5′ UTR was probed using SHAPE technique. These results identified a very stable secondary structure for both 5′and 3′ UTRs. Primer extension inhibition (toe‐printing) was used to identify the initial 43S ribosome binding site. These results suggest a novel mechanism for 43S ribosome binding to the 3′BTE followed by subsequent transfer to the 5′end.

Cation-dependent folding of 3′ cap-independent translation elements facilitates interaction of a 17-nucleotide conserved sequence with eIF4G

The 3′-untranslated regions of many plant viral RNAs contain cap-independent translation elements (CITEs) that drive translation initiation at the 5′-end of the mRNA. The barley yellow dwarf virus-like CITE (BTE) stimulates translation by binding the eIF4G subunit of translation initiation factor eIF4F with high affinity. To understand this interaction, we characterized the dynamic structural properties of the BTE, mapped the eIF4G-binding sites on the BTE and identified a region of eIF4G that is crucial for BTE binding. BTE folding involves cooperative uptake of magnesium ions and is driven primarily by charge neutralization. Footprinting experiments revealed that functional eIF4G fragments protect the highly conserved stem–loop I and a downstream bulge. The BTE forms a functional structure in the absence of protein, and the loop that base pairs the 5′-untranslated region (5′-UTR) remains solvent-accessible at high eIF4G concentrations. The region in eIF4G between the eIF4E-binding site and the MIF4G region is required for BTE binding and translation. The data support the model in which the eIF4F complex binds directly to the BTE which base pairs simultaneously to the 5′-UTR, allowing eIF4F to recruit the 40S ribosomal subunit to the 5′-end.

Untranslated regions of diverse plant viral RNAs vary greatly in translation enhancement efficiency

BackgroundWhole plants or plant cell cultures can serve as low cost bioreactors to produce massive amounts of a specific protein for pharmacological or industrial use. To maximize protein expression, translation of mRNA must be optimized. Many plant viral RNAs harbor extremely efficient translation enhancers. However, few of these different translation elements have been compared side-by-side. Thus, it is unclear which are the most efficient translation enhancers. Here, we compare the effects of untranslated regions (UTRs) containing translation elements from six plant viruses on translation in wheat germ extract and in monocotyledenous and dicotyledenous plant cells.ResultsThe highest expressing uncapped mRNAs contained viral UTRs harboring Barley yellow dwarf virus (BYDV)-like cap-independent translation elements (BTEs). The BYDV BTE conferred the most efficient translation of a luciferase reporter in wheat germ extract and oat protoplasts, while uncapped mRNA containing the BTE from Tobacco necrosis virus-D translated most efficiently in tobacco cells. Capped mRNA containing the Tobacco mosaic virus omega sequence was the most efficient mRNA in tobacco cells. UTRs from Satellite tobacco necrosis virus, Tomato bushy stunt virus, and Crucifer-infecting tobamovirus (crTMV) did not stimulate translation efficiently. mRNA with the crTMV 5′ UTR was unstable in tobacco protoplasts.ConclusionsBTEs confer the highest levels of translation of uncapped mRNAs in vitro and in vivo, while the capped omega sequence is most efficient in tobacco cells. These results provide a basis for understanding mechanisms of translation enhancement, and for maximizing protein synthesis in cell-free systems, transgenic plants, or in viral expression vectors.

A long-distance RNA–RNA interaction plays an important role in programmed − 1 ribosomal frameshifting in the translation of p88 replicase protein of Red clover necrotic mosaic virus

Programmed − 1 ribosomal frameshifting (− 1 PRF) is one viral translation strategy to express overlapping genes in positive-strand RNA viruses. Red clover necrotic mosaic virus (RCNMV) uses this strategy to express its replicase component protein p88. In this study, we used a cell-free translation system to map cis-acting RNA elements required for − 1 PRF. Our results show that a small stem-loop structure adjacent to the cap-independent translation element in the 3′ untranslated region (UTR) of RCNMV RNA1 is required for − 1 PRF. Site-directed mutagenesis experiments suggested that this stem-loop regulates − 1 PRF via base-pairing with complementary sequences in a bulged stem-loop adjacent to the shifty site. The existence of RNA elements responsible for − 1 PRF and the cap-independent translation of replicase proteins in the 3′ UTR of RNA1 might be important for switching translation to replication and for regulating the ratio of p88 to p27.