scholarly journals A novel type of IRES having variable numbers of eIF4E-binding site and synergistic effect in RNA2 of WYMV isolates

2021 ◽  
Author(s):  
Xuefeng Yuan ◽  
Guowei Geng ◽  
Chengming Yu ◽  
Xiangdong Li ◽  
Kerong Shi
Keyword(s):  
Synergistic Effect ◽  
Binding Site ◽  
Rna Structure ◽  
Yellow Mosaic Virus ◽  
Wheat Yellow Mosaic Virus ◽  
Entry Site ◽  
Independent Manner ◽  
Internal Ribosome Entry ◽  
Structure Probing ◽  
Evolution Pattern

Some viral proteins were translated in cap-independent manner via internal ribosome entry site (IRES), which ever maintained conservative characteristic among different isolates of same species of virus. However, IRES activity presented 7-fold of variance in RNA2 of wheat yellow mosaic virus (WYMV) HC and LYJN isolates. Based on RNA structure probing and mutagenesis assay, the loosened middle stem of H1 and hepta-nucleotide top loop of H2 in LYJN isolate synergistically ensured the higher IRES activity than that in HC isolate. In addition, the conserved top loop of H1 ensured basic IRES activity in HC and LYJN isolates. RNA2 5′-UTR specifically interacted with the wheat eIF4E, which was accomplished by the top loop of H1 in HC isolate or the top loop of H1 and H2 in LYJN isolate. Different IRES activity of WYMV RNA2 was regulated by different numbers of eIF4E-binding site and their synergistic effect, which was accomplished by the proximity of H1 and H2 due to the flexibility of middle stem in H1. It is represented a novel evolution pattern of IRES.

scholarly journals Conditional Rather than Absolute Requirements of the Capsid Coding Sequence for Initiation of Methionine-Independent Translation in Plautia stali Intestine Virus

2003 ◽  
Vol 77 (22) ◽  
pp. 12002-12010 ◽  
Author(s):  
Norihiro Shibuya ◽  
Takashi Nishiyama ◽  
Yasushi Kanamori ◽  
Hitoshi Saito ◽  
Nobuhiko Nakashima
Keyword(s):  
Rna Structure ◽  
Tertiary Structure ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Reading Frame ◽  
Structure Probing ◽  
Absolute Requirement ◽  
Plautia Stali ◽  
Independent Translation

ABSTRACT The positive-stranded RNA genome of Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) in an intergenic region (IGR). The IGR-IRES of PSIV initiates translation of the capsid protein by using CAA, the codon for glutamine. It was previously reported (J. Sasaki and N. Nakashima, J. Virol. 73:1219-1226, 1999) that IGR-IRES extended by several nucleotides into the capsid open reading frame (ORF). Despite the fact that the secondary structure model of the IGR-IRES is highly conserved, we were unable to find structural similarities in the 5′ region of the capsid ORFs in related viruses. Therefore, we reevaluated the role of the capsid ORF in IGR-IRES-mediated translation in PSIV. Mutation of the CAA codon with various triplets did not inhibit IGR-IRES-mediated translation. N-terminal amino acid analyses of mutated products showed that the IGR-IRES could initiate translation by using various elongator tRNAs. By replacement of the capsid ORF with exogenous coding sequences having AUG deleted, translation products were produced in most cases, but capsid-exogenous fusion proteins were produced more efficiently than were the translation products. These data indicate that the 5′ part of the capsid ORF is not an absolute requirement for the IGR-IRES-mediated translation. RNA structure probing analyses showed that the 5′ part of the capsid ORF was a single strand, while that of exogenous reading frames was structured. Exogenous sequences also caused structural distortion in the 3′ part of the IGR-IRES. We hypothesize that the single-stranded capsid ORF helps to form the tertiary structure of the IGR-IRES and facilitates precise positioning of ribosomes.

scholarly journals BDNF-induced local translation of GluA1 is regulated by HNRNP A2/B1

2020 ◽  
Vol 6 (47) ◽  
pp. eabd2163
Author(s):  
Youngseob Jung ◽  
Ji-Young Seo ◽  
Hye Guk Ryu ◽  
Do-Yeon Kim ◽  
Kyung-Ha Lee ◽  
...  
Keyword(s):  
Ampa Receptor ◽  
Molecular Mechanisms ◽  
Receptor Expression ◽  
Spine Density ◽  
Entry Site ◽  
Independent Manner ◽  
Internal Ribosome Entry ◽  
Ampa Receptor Subunit ◽  
Internal Initiation ◽  
Underlying Mechanisms

The AMPA receptor subunit GluA1 is essential for induction of synaptic plasticity. While various regulatory mechanisms of AMPA receptor expression have been identified, the underlying mechanisms of GluA1 protein synthesis are not fully understood. In neurons, axonal and dendritic mRNAs have been reported to be translated in a cap-independent manner. However, molecular mechanisms of cap-independent translation of synaptic mRNAs remain largely unknown. Here, we show that GluA1 mRNA contains an internal ribosome entry site (IRES) in the 5′UTR. We also demonstrate that heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 interacts with GluA1 mRNA and mediates internal initiation of GluA1. Brain-derived neurotrophic factor (BDNF) stimulation increases IRES-mediated GluA1 translation via up-regulation of HNRNP A2/B1. Moreover, BDNF-induced GluA1 expression and dendritic spine density were significantly decreased in neurons lacking hnRNP A2/B1. Together, our data demonstrate that IRES-mediated translation of GluA1 mRNA is a previously unidentified feature of local expression of the AMPA receptor.

scholarly journals A unique internal ribosome entry site representing a dynamic equilibrium state of RNA tertiary structure in the 5′-UTR of Wheat yellow mosaic virus RNA1

2019 ◽  
Author(s):  
Guowei Geng ◽  
Chengming Yu ◽  
Xiangdong Li ◽  
Xuefeng Yuan
Keyword(s):  
Equilibrium State ◽  
Mosaic Virus ◽  
Dynamic Equilibrium ◽  
Yellow Mosaic Virus ◽  
Wheat Yellow Mosaic Virus ◽  
Long Distance ◽  
Internal Ribosome Entry ◽  
Interaction Sites ◽  
Dynamic Equilibrium State ◽  
Rna Interaction

Abstract Internal ribosome entry sites (IRESes) were first reported in RNA viruses and subsequently identified in cellular mRNAs. In this study, IRES activity of the 5′-UTR in Wheat yellow mosaic virus (WYMV) RNA1 was identified, and the 3′-UTR synergistically enhanced this IRES activity via long-distance RNA–RNA interaction between C80U81and A7574G7575. Within the 5′-UTR, the hairpin 1(H1), flexible hairpin 2 (H2) and linker region (LR1) between H1 and H2 played an essential role in cap-independent translation, which is associated with the structural stability of H1, length of discontinuous stems and nucleotide specificity of the H2 upper loop and the long-distance RNA–RNA interaction sites in LR1. The H2 upper loop is a target region of the eIF4E. Cytosines (C55, C66, C105 and C108) in H1 and H2 and guanines (G73, G79 and G85) in LR1 form discontinuous and alternative base pairing to maintain the dynamic equilibrium state, which is used to elaborately regulate translation at a suitable level. The WYMV RNA1 5′-UTR contains a novel IRES, which is different from reported IRESes because of the dynamic equilibrium state. It is also suggested that robustness not at the maximum level of translation is the selection target during evolution of WYMV RNA1.

scholarly journals The Ribosome Binding Site of Hepatitis C Virus mRNA

2001 ◽  
Vol 75 (16) ◽  
pp. 7629-7636 ◽  
Author(s):  
J. Robin Lytle ◽  
Lily Wu ◽  
Hugh D. Robertson
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Binding Site ◽  
Ribosome Binding Site ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Promising Target ◽  
Hcv Ires

ABSTRACT Hepatitis C virus (HCV) infects an estimated 170 million people worldwide, the majority of whom develop a chronic infection which can lead to severe liver disease, and for which no generally effective treatment yet exists. A promising target for treatment is the internal ribosome entry site (IRES) of HCV, a highly conserved domain within a highly variable RNA. Never before have the ribosome binding sites of any IRES domains, cellular or viral, been directly characterized. Here, we reveal that the HCV IRES sequences most closely associated with 80S ribosomes during protein synthesis initiation are a series of discontinuous domains together comprising by far the largest ribosome binding site yet discovered.

scholarly journals Roles of the 5′ Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Tomohisa Tanaka ◽  
Teruhime Otoguro ◽  
Atsuya Yamashita ◽  
Hirotake Kasai ◽  
Takasuke Fukuhara ◽  
...  
Keyword(s):  
Viral Replication ◽  
Internal Ribosome Entry Site ◽  
Untranslated Region ◽  
Rna Stability ◽  
Specific Region ◽  
Entry Site ◽  
Subgenomic Replicon ◽  
Independent Manner ◽  
Internal Ribosome Entry ◽  
Domain I

ABSTRACTThe 5′ untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5′ UTR consisting of a large 5′-terminal domain (I); three additional domains (I′, II, and III), which are homologous to domains I, II, and III, respectively, of HCV; and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5′ UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5′ UTR was lower than that of the HCV 5′ UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5′ UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domains I and I′ of EHcV (the 5′-proximal EHcV-specific region) improved RNA stability and provided the HCV SGR with microRNA 122 (miR-122)-independent replication capability, while EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domains I and I′ improves RNA stability and viral replication regardless of miR-122 expression. The 5′-proximal EHcV-specific region may represent an inherent mechanism to facilitate viral replication in nonhepatic tissues.IMPORTANCEEHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs in horses, donkeys, and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5′ untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5′-proximal EHcV-specific region enhances viral replication and RNA stability in a miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5′ UTR contributes to the differences in tissue tropism observed between these hepaciviruses.

scholarly journals Deconstructing internal ribosome entry site elements: an update of structural motifs and functional divergences

Open Biology ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 180155 ◽  
Author(s):  
Gloria Lozano ◽  
Rosario Francisco-Velilla ◽  
Encarnacion Martinez-Salas
Keyword(s):  
Internal Ribosome Entry Site ◽  
Rna Structure ◽  
Rna Binding ◽  
Building Blocks ◽  
Regulatory Elements ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ribonucleoprotein Complexes ◽  
Structure Conservation ◽  
Initiation Of Translation

Beyond the general cap-dependent translation initiation, eukaryotic organisms use alternative mechanisms to initiate protein synthesis. Internal ribosome entry site (IRES) elements are cis -acting RNA regions that promote internal initiation of translation using a cap-independent mechanism. However, their lack of primary sequence and secondary RNA structure conservation, as well as the diversity of host factor requirement to recruit the ribosomal subunits, suggest distinct types of IRES elements. In spite of this heterogeneity, conserved motifs preserve sequences impacting on RNA structure and RNA–protein interactions important for IRES-driven translation. This conservation brings the question of whether IRES elements could consist of basic building blocks, which upon evolutionary selection result in functional elements with different properties. Although RNA-binding proteins (RBPs) perform a crucial role in the assembly of ribonucleoprotein complexes, the versatility and plasticity of RNA molecules, together with their high flexibility and dynamism, determines formation of macromolecular complexes in response to different signals. These properties rely on the presence of short RNA motifs, which operate as modular entities, and suggest that decomposition of IRES elements in short modules could help to understand the different mechanisms driven by these regulatory elements. Here we will review evidence suggesting that model IRES elements consist of the combination of short modules, providing sites of interaction for ribosome subunits, eIFs and RBPs, with implications for definition of criteria to identify novel IRES-like elements genome wide.

scholarly journals Trans-Activator Binding Site Context in RCNMV Modulates Subgenomic mRNA Transcription

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2252
Author(s):  
Jennifer S. H. Im ◽  
Laura R. Newburn ◽  
Gregory Kent ◽  
K. Andrew White
Keyword(s):  
Binding Site ◽  
Rna Structure ◽  
Red Clover ◽  
Mrna Levels ◽  
Mrna Transcription ◽  
Base Pairing ◽  
Independent Manner ◽  
The Impact

Many positive-sense RNA viruses transcribe subgenomic (sg) mRNAs during infections that template the translation of a subset of viral proteins. Red clover necrotic mosaic virus (RCNMV) expresses its capsid protein through the transcription of a sg mRNA from RNA1 genome segment. This transcription event is activated by an RNA structure formed by base pairing between a trans-activator (TA) in RNA2 and a trans-activator binding site (TABS) in RNA1. In this study, the impact of the structural context of the TABS in RNA1 on the TA–TABS interaction and sg mRNA transcription was investigated using in vitro and in vivo approaches. The results (i) generated RNA secondary structure models for the TA and TABS, (ii) revealed that the TABS is partially base paired with proximal upstream sequences, which limits TA access, (iii) demonstrated that the aforementioned intra-RNA1 base pairing involving the TABS modulates the TA–TABS interaction in vitro and sg mRNA levels during infections, and (iv) revealed that the TABS in RNA1 can be modified to mediate sg mRNA transcription in a TA-independent manner. These findings advance our understanding of transcriptional regulation in RCNMV and provide novel insights into the origin of the TA–TABS interaction.

scholarly journals Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements

2001 ◽  
Vol 21 (8) ◽  
pp. 2826-2837 ◽  
Author(s):  
Arun Venkatesan ◽  
Asim Dasgupta
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Independent Manner ◽  
Internal Ribosome Entry

ABSTRACT We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.

scholarly journals EZH2 co-opts gain-of-function p53 mutants to promote cancer growth and metastasis

2018 ◽  
Author(s):  
Yu Zhao ◽  
Liya Ding ◽  
Dejie Wang ◽  
Zhenqing Ye ◽  
Yunqian Pan ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
P53 Protein ◽  
Protein Translation ◽  
Cancer Growth ◽  
Common Mechanism ◽  
Entry Site ◽  
Gain Of Function ◽  
Independent Manner ◽  
Internal Ribosome Entry ◽  
P53 Mrna

AbstractWith the unfolding of more and more cancer-driven gain-of-function (GOF) mutants of p53, it is important to define a common mechanism to systematically target different mutants rather than develop strategies tailored to inhibit each mutant individually. Here, using RNA immunoprecipitation sequencing (RIP-seq) we identified EZH2 as a p53 mRNA-binding protein. EZH2 bound to the internal ribosome entry site (IRES) in the 5’ untranslated region (5’UTR) of p53 mRNA and enhanced p53 protein translation in a methyltransferase-independent manner. EZH2 augmented p53 GOF mutant-mediated cancer growth and metastasis by increasing p53 GOF mutant protein level. EZH2 overexpression associated with the worse outcome only in patients with p53-mutated cancer. Depletion of EZH2 by antisense oligonucleotides inhibited p53 GOF mutant-mediated cancer growth. Our findings reveal a non-methyltransferase function of EZH2 that controls protein translation of p53 GOF mutants, inhibition of which causes synthetic lethality in cancer cells expressing p53 GOF mutants.

scholarly journals The RNA encoding the microtubule-associated protein tau has extensive structure that affects its biology

2019 ◽  
Author(s):  
Jonathan L. Chen ◽  
Walter N. Moss ◽  
Adam Spencer ◽  
Peiyuan Zhang ◽  
Jessica L. Childs-Disney ◽  
...  
Keyword(s):  
Rna Structure ◽  
Protein Production ◽  
Regulatory Elements ◽  
Therapeutic Benefit ◽  
Untranslated Regions ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Disease Mechanisms ◽  
Exon 10

ABSTRACTTauopathies are neurodegenerative diseases that affect millions of people worldwide including those with Alzheimer’s disease. While many efforts have focused on understanding the role of tau protein in neurodegeneration, there has been little done to systematically analyze and study the structures within tau’s encoding RNA and their connection to disease pathology. Knowledge of RNA structure can provide insights into disease mechanisms and how to affect protein production for therapeutic benefit. Using computational methods based on thermodynamic stability and evolutionary conservation, we identified structures throughout the tau pre-mRNA, especially at exon-intron junctions and within the 5′ and 3′ untranslated regions (UTRs). In particular, structures were identified at twenty exon-intron junctions. The 5′ UTR contains one structured region, which lies within a known internal ribosome entry site. The 3′ UTR contains eight structured regions, including one that contains a polyadenylation signal. A series of functional experiments were carried out to assess the effects of mutations associated with mis-regulation of alternative splicing of exon 10 and to identify regions of the 3′ UTR that contain cis-regulatory elements. These studies defined novel structural regions within the mRNA that affect stability and pre-mRNA splicing and may lead to new therapeutic targets for treating tau-associated diseases.

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