cis-Acting Elements of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

Witherell, Gary W.; Schultz-Witherell, Carla S.; Wimmer, Eckard

doi:10.1006/viro.1995.0081

Cited by 35 publications

(29 citation statements)

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“…Deletion ⌬J5 reduced IRES-mediated translation to 15% of the level of the wild-type IRES (lanes 5 and 6), and translation was synergistically impaired by combining deletions ⌬J5 and ⌬K2Ј (compare lanes 7 and 8 with lanes 3 and 4 and lanes 5 and 6). These results confirm the importance of J5 and K2 hairpins for IRES function (13,32,48). Binding of eIF4G and eIF4F to IRESs containing deletions in the J-K domain.…”

Section: Vol 23 2003 Initiation Factor-mediated Change In Ires Confsupporting

confidence: 79%

“…Although the J-K domain is essential, it is by itself not sufficient for EMCV IRES function, and experiments to date account only for the function of the central core of this domain. The extensive sequence conservation in EMCV-like IRESs extends over much of the J-K domain (18), including regions such as the terminal loops of the J and K domains and the J3 helix that have been found to be important for initiation (13,32,48). The adjacent domain I also plays an essential but as yet undefined role in EMCV IRES function, and the flanking domain H, domain L, and sequences downstream of domain L all also play accessory roles (7,9,18,20).…”

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confidence: 99%

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Eukaryotic Initiation Factors 4G and 4A Mediate Conformational Changes Downstream of the Initiation Codon of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

Kolupaeva

Lomakin

Pestova

et al. 2003

Molecular and Cellular Biology

113

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Initiation of translation of encephalomyocarditis virus mRNA is mediated by an internal ribosome entry site (IRES) comprising structural domains H, I, J-K, and L immediately upstream of the initiation codon AUG at nucleotide 834 (AUG 834 ). Assembly of 48S ribosomal complexes on the IRES requires eukaryotic initiation factor 2 (eIF2), eIF3, eIF4A, and the central domain of eIF4G to which eIF4A binds. Footprinting experiments confirmed that eIF4G binds a three-way helical junction in the J-K domain and showed that it interacts extensively with RNA duplexes in the J-K and L domains. Deletion of apical hairpins in the J and K domains synergistically impaired the binding of eIF4G and IRES function. Directed hydroxyl radical probing, done by using Fe(II) tethered to surface residues in eIF4G's central domain, indicated that it is oriented with its N terminus towards the base of domain J and its C terminus towards the apex. eIF4G recruits eIF4A to a defined location on the IRES, and the eIF4G/eIF4A complex caused localized ATP-independent conformational changes in the eIF4G-binding region of the IRES. This complex also induced more extensive conformational rearrangements at the 3 border of the ribosome binding site that required ATP and active eIF4A. We propose that these conformational changes prepare the region flanking AUG 834 for productive binding of the ribosome.Translation of a subset of cellular and viral mRNAs is initiated by end-independent binding of ribosomes to an internal ribosome entry site (IRES) in the 5Ј untranslated region. IRESs were first identified in different picornavirus RNA genomes, and these IRESs are now classified into two major groups, one of which includes those of the encephalomyocarditis virus (EMCV), Theiler's murine encephalitis virus (TMEV), and foot-and-mouth disease virus (FMDV) (17). The EMCV IRES is ϳ450 nucleotides (nt) long and comprises H, I, J-K, and L structural domains upstream of the initiation codon AUG at nucleotide 834 (AUG 834 ). Ribosomal initiation complexes attach directly to AUG 834 , and initiation does not involve scanning (19,35). Recent analysis has begun to resolve molecular details of the mechanism of initiation on EMCVlike IRESs. Viral and cellular IRES-containing mRNAs can be translated under conditions when the cap-mediated mode of end-dependent initiation that is used by most mRNAs is inhibited (12), and studies of initiation on viral IRESs are therefore yielding insights into mechanisms that enable selective translation of mRNAs in the cell.The stage that differentiates IRES-mediated initiation from the conventional cap-mediated mode of initiation is the mechanism by which the 43S ribosomal preinitiation complex engages mRNA to form a 48S complex at the initiation codon (16,36). The 43S complex consists of a 40S ribosomal subunit, initiator tRNA, and eukaryotic initiation factors (eIFs), including eIF2 and eIF3. In the process of cap-mediated initiation, eIF4F is thought to create a binding site for this complex adjacent to the 5Ј-terminal m 7 G cap by ...

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Section: Vol 23 2003 Initiation Factor-mediated Change In Ires Confsupporting

confidence: 79%

mentioning

confidence: 99%

Eukaryotic Initiation Factors 4G and 4A Mediate Conformational Changes Downstream of the Initiation Codon of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

Kolupaeva

Lomakin

Pestova

et al. 2003

Molecular and Cellular Biology

113

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“…PTBP1 serves as a repressor of alternative splicing in mammalian cells (24)(25)(26)(27)(28)(29) and contains RNA-binding domains, each of which binds to CU-rich elements (30). It is involved in polyadenylation of the pre-mRNA 3 0 end (31-33) and also plays an important role in translational regulation of a subset of RNA transcription through internal ribosome entry sites (21,(34)(35)(36)(37). As for malignancies, PTBP1 is overexpressed in glioblastoma (7,8) and breast cancer (38), suppresses p27 expression, and contributes largely to cell proliferation (21,39).…”

Section: Discussionmentioning

confidence: 99%

Significance of Polypyrimidine Tract–Binding Protein 1 Expression in Colorectal Cancer

Takahashi

Nishimura

Kagawa

et al. 2015

Molecular Cancer Therapeutics

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Polypyrimidine tract-binding protein (PTBP1) is an RNAbinding protein with various molecular functions related to RNA metabolism and a major repressive regulator of alternative splicing, causing exon skipping in numerous alternatively spliced pre-mRNAs. Here, we have investigated the role of PTBP1 in colorectal cancer. PTBP1 expression levels were significantly overexpressed in cancerous tissues compared with corresponding normal mucosal tissues. We also observed that PTBP1 expression levels, c-MYC expression levels, and PKM2: PKM1 ratio were positively correlated in colorectal cancer specimens. Moreover, PTBP1 expression levels were positively correlated to poor prognosis and lymph node metastasis. In analyses of colorectal cancer cells using siRNA for PTBP1, we observed that PTBP1 affects cell invasion, which was partially correlated to CD44 splicing, and this correlation was also confirmed in clinical samples. PTBP1 expression also affected anchorage-independent growth in colorectal cancer cell lines. PTBP1 expression also affected cell proliferation. Using timelapse imaging analysis, PTBP1 was implicated in prolonged G 2 -M phase in HCT116 cells. As for the mechanism of prolonged G 2 -M phase in HCT116 siPTBP1 cells, Western blotting revealed that PTBP1 expression level was correlated to CDK11 p58 expression level, which was reported to play an important role on progression to complete mitosis. These findings indicated that PTBP1 is a potential therapeutic target for colorectal cancer.

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“…These structural features are particularly well conserved between cardio/aphthoviruses and parechoviruses, consistent with their importance to the IRES core. Domains I, J, and K also appear to be the most critical for translation initiation in cardio/aphthoviruses (13,25,74). Although translation in picornaviruses may require other regions of the 5ЈUTR participating in a subtle interplay between translation and RNA replication (16), the structural domains defined here seem to represent the irreducible minimum for IRES activity.…”

Section: Discussionmentioning

confidence: 94%

“…Much of the picornavirus 5ЈUTR makes up the internal ribosome entry site (IRES), needed for the cap-independent translation exhibited by these viruses. In vitro and in vivo translation has been used to define IRES activity for representatives of the Aphthovirus (3,43), Cardiovirus (13,34,35,74), Enterovirus (22,50,55), Hepatovirus (6,7,18), and Rhinovirus (29,71) genera. An IRES element also occurs in the genome of the hepatitis C virus and in some cellular mRNAs (45,48).…”

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confidence: 99%

In Vivo and In Vitro Identification of Structural and Sequence Elements of the Human Parechovirus 5′ Untranslated Region Required for Internal Initiation

Nateri

Hughes

Stanway

2000

J Virol

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Sequence analysis of the picornavirus echovirus 22 led to its classification as the first member of a new genus, Parechovirus, and renaming as human parechovirus type 1 (HPeV1). Although distinct from other genera in most of the genome, the 5 untranslated region (5UTR) shows similarities to that of cardio/ aphthoviruses in some of its structural domains (A to L). The 5UTR plays an important role in picornavirus translation initiation and in RNA synthesis. To investigate translation in HPeV1, we engineered an extensive range of mutations (including precise deletions and point mutations) into the 5UTR. Their effects were studied both by in vitro transcription-translation using a bicistronic construct and by in vivo studies using an infectious, full-length HPeV1 cDNA. These approaches allowed the HPeV1 internal ribosome entry site (IRES) to be mapped. Deletions within the first 298 nucleotides had little impact in the in vitro system, while deletions of nucleotides 298 to 538 had a significant effect. Precise removal of domains H and L (nucleotides 287 to 316 and 664 to 682, respectively) did not significantly reduce translation efficiency in vitro, while domains I, J, and K (nucleotides 327 to 545, 551 to 661, and 614 to 645, respectively) appeared to have much more important roles. Mutation of a phylogenetically conserved GNRA motif (positions 421 to 424) within domain I severely reduced translation. We also confirmed the identity of the AUG (positions 710 to 712) which initiates the open reading frame, the positive identification of which has not been possible previously, as the N terminus of the polyprotein is blocked and not amenable to sequence analysis. This is therefore important in understanding parechovirus genome organization. Mutation of the AUG or an upstream polypyrimidine tract leads to aberrant translation, suggesting they both form part of the parechovirus Yn-Xm-AUG motif. In vivo experiments confirmed the importance of domains I, J, and K, the conserved GNRA motif, polypyrimidine sequences, and AUG, as mutations here were lethal. These features are also important in the IRES elements of cardio/ aphthoviruses, but other features reported to be part of the IRES of some members of these genera, notably domains H and L, do not appear to be critical in HPeV1. This adds weight to the idea that there may be functional differences between the IRES elements of different picornaviruses, even when they share significant structural similarity.Picornaviridae is a large family of single-stranded positivesense RNA viruses, which includes a number of important human and animal pathogens. Until recently, picornaviruses were classified into five genera, Aphthovirus, Cardiovirus, Enterovirus, Hepatovirus, and Rhinovirus, but two former enteroviruses (echovirus 22 and 23) have now been placed in a new genus, Parechovirus, and renamed human parechovirus types 1 and 2 (HPeV1 and -2), respectively (68). Human parechoviruses had previously been shown to exhibit several atypical properties, including unusual cytopathology...

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cis-Acting Elements of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

Cited by 35 publications

References 0 publications

Eukaryotic Initiation Factors 4G and 4A Mediate Conformational Changes Downstream of the Initiation Codon of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

Eukaryotic Initiation Factors 4G and 4A Mediate Conformational Changes Downstream of the Initiation Codon of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

Significance of Polypyrimidine Tract–Binding Protein 1 Expression in Colorectal Cancer

In Vivo and In Vitro Identification of Structural and Sequence Elements of the Human Parechovirus 5′ Untranslated Region Required for Internal Initiation

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