scholarly journals Regulation of Gene Expression by Internal Ribosome Entry Sites or Cryptic Promoters: the eIF4G Story

2002 ◽  
Vol 22 (21) ◽  
pp. 7372-7384 ◽  
Author(s):  
Baoguang Han ◽  
Jian-Ting Zhang
Keyword(s):  
Northern Blot ◽  
Regulation Of Gene Expression ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cryptic Promoter ◽  
Internal Ribosome Entry ◽  
Internal Promoter ◽  
Internal Ribosome Entry Sites ◽  
Cellular Mrnas ◽  
Factor C

ABSTRACT As an alternative to the scanning mechanism of initiation, the direct-internal-initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5′ untranslated region (5′-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5′-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5′-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBPβ. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5′-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5′-UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove useful in future studies to examine more rigorously the claim that there is IRES activity in cellular mRNAs.

scholarly journals Unlike for cellular mRNAs and other viral internal ribosome entry sites (IRESs), the eIF3 subunit e is not required for the translational activity of the HCV IRES

2020 ◽  
Vol 295 (7) ◽  
pp. 1843-1856
Author(s):  
Baptiste Panthu ◽  
Solène Denolly ◽  
Cendrine Faivre-Moskalenko ◽  
Théophile Ohlmann ◽  
François-Loïc Cosset ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Hcv Rna ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Sites ◽  
Subunit E ◽  
Cellular Mrnas ◽  
Hcv Ires

Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5′UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels.

scholarly journals A novel Bcr-Abl–mTOR–eIF4A axis regulates IRES-mediated translation of LEF-1

Open Biology ◽  
2014 ◽  
Vol 4 (11) ◽  
pp. 140180 ◽  
Author(s):  
Becky Pinjou Tsai ◽  
Judith Jimenez ◽  
Sharon Lim ◽  
Kerry D. Fitzgerald ◽  
Min Zhang ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Cell Transformation ◽  
Mammalian Target Of Rapamycin ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Combination Drug ◽  
Internal Ribosome Entry ◽  
Cellular Mrnas ◽  
Translation Mechanism ◽  
Oncogenic Protein

Internal ribosome entry sites (IRESs) in cellular mRNAs direct expression of growth-promoting factors through an alternative translation mechanism that has yet to be fully defined. Lymphoid enhancer factor-1 (LEF-1), a Wnt-mediating transcription factor important for cell survival and metastasis in cancer, is produced via IRES-directed translation, and its mRNA is frequently upregulated in malignancies, including chronic myeloid leukaemia (CML). In this study, we determined that LEF1 expression is regulated by Bcr-Abl, the oncogenic protein that drives haematopoietic cell transformation to CML. We have previously shown that the LEF1 5′ untranslated region recruits a complex of proteins to its IRES, including the translation initiation factor eIF4A. In this report, we use two small molecule inhibitors, PP242 (dual mTOR (mammalian target of rapamycin) kinase inhibitor) and hippuristanol (eIF4A inhibitor), to define IRES regulation via a Bcr-Abl–mTOR–eIF4A axis in CML cell lines and primary patient leukaemias. We found that LEF1 and other IRESs are uniquely sensitive to the activities of Bcr-Abl/mTOR. Most notably, we discovered that eIF4A, an RNA helicase, elicits potent non-canonical effects on the LEF1 IRES. Hippuristanol inhibition of eIF4A stalls translation of IRES mRNA and triggers dissociation from polyribosomes. We propose that a combination drug strategy which targets mTOR and IRES-driven translation disrupts key factors that contribute to growth and proliferation in CML.

scholarly journals Importance of Arginine 20 of the Swine Vesicular Disease Virus 2A Protease for Activity and Virulence

2005 ◽  
Vol 79 (1) ◽  
pp. 428-440 ◽  
Author(s):  
Toru Inoue ◽  
Soren Alexandersen ◽  
Angela T. Clark ◽  
Ciara Murphy ◽  
Melvyn Quan ◽  
...  
Keyword(s):  
Disease Virus ◽  
Direct Sequencing ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Internal Ribosome Entry ◽  
Virus Growth ◽  
Swine Vesicular Disease Virus ◽  
2A Protease ◽  
Viral Polyprotein ◽  
Vesicular Disease

ABSTRACT A major virulence determinant of swine vesicular disease virus (SVDV), an Enterovirus that causes an acute vesicular disease, has been mapped to residue 20 of the 2A protease. The SVDV 2A protease cleaves the 1D-2A junction in the viral polyprotein, induces cleavage of translation initiation factor eIF4GI, and stimulates the activity of enterovirus internal ribosome entry sites (IRESs). The 2A protease from an attenuated strain of SVDV (Ile at residue 20) is significantly defective at inducing cleavage of eIF4GI and the activation of IRES-dependent translation compared to the 2A protease from a pathogenic strain (J1/73, Arg at residue 20), but the two proteases have similar 1D-2A cleavage activities (Y. Sakoda, N. Ross-Smith, T. Inoue, and G. J. Belsham, J. Virol. 75:10643-10650, 2001). Residue 20 has now been modified to every possible amino acid, and the activities of each mutant 2A protease has been analyzed. Selected mutants were reconstructed into full-length SVDV cDNA, and viruses were rescued. The rate of virus growth in cultured swine kidney cells reflected the efficiency of 2A protease activity. In experimentally infected pigs, all four of the mutant viruses tested displayed much-reduced virulence compared to the J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was detected. Direct sequencing of cDNA derived from samples taken early and late in infection indicated that a gradual selection-reversion to a more efficient protease occurred. The data indicated that extensive sequence change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease.

scholarly journals Focus on Translation Initiation of the HIV-1 mRNAs

2018 ◽  
Vol 20 (1) ◽  
pp. 101 ◽  
Author(s):  
Sylvain de Breyne ◽  
Théophile Ohlmann
Keyword(s):  
Translation Initiation ◽  
Translational Control ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cellular Proteins ◽  
Internal Ribosome Entry ◽  
Eukaryotic Translation ◽  
Cellular Environment ◽  
Key Steps ◽  
Hiv 1

To replicate and disseminate, viruses need to manipulate and modify the cellular machinery for their own benefit. We are interested in translation, which is one of the key steps of gene expression and viruses that have developed several strategies to hijack the ribosomal complex. The type 1 human immunodeficiency virus is a good paradigm to understand the great diversity of translational control. Indeed, scanning, leaky scanning, internal ribosome entry sites, and adenosine methylation are used by ribosomes to translate spliced and unspliced HIV-1 mRNAs, and some require specific cellular factors, such as the DDX3 helicase, that mediate mRNA export and translation. In addition, some viral and cellular proteins, including the HIV-1 Tat protein, also regulate protein synthesis through targeting the protein kinase PKR, which once activated, is able to phosphorylate the eukaryotic translation initiation factor eIF2α, which results in the inhibition of cellular mRNAs translation. Finally, the infection alters the integrity of several cellular proteins, including initiation factors, that directly or indirectly regulates translation events. In this review, we will provide a global overview of the current situation of how the HIV-1 mRNAs interact with the host cellular environment to produce viral proteins.

scholarly journals PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes

2003 ◽  
Vol 84 (12) ◽  
pp. 3263-3274 ◽  
Author(s):  
Idoia Burgui ◽  
Tomás Aragón ◽  
Juan Ortín ◽  
Amelia Nieto
Keyword(s):  
Influenza Virus ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Published Data ◽  
Ns1 Protein ◽  
Viral Mrnas ◽  
Independent Manner ◽  
Cellular Mrnas

It has previously been shown that influenza virus NS1 protein enhances the translation of viral but not cellular mRNAs. This enhancement occurs by increasing the rate of translation initiation and requires the 5′UTR sequence, common to all viral mRNAs. In agreement with these findings, we show here that viral mRNAs, but not cellular mRNAs, are associated with NS1 during virus infection. We have previously reported that NS1 interacts with the translation initiation factor eIF4GI, next to its poly(A)-binding protein 1 (PABP1)-interacting domain and that NS1 and eIF4GI are associated in influenza virus-infected cells. Here we show that NS1, although capable of binding poly(A), does not compete with PABP1 for association with eIF4GI and, furthermore, that NS1 and PABP1 interact both in vivo and in vitro in an RNA-independent manner. The interaction maps between residues 365 and 535 in PABP1 and between residues 1 and 81 in NS1. These mapping studies, together with those previously reported for NS1–eIF4GI and PABP1–eIF4GI interactions, imply that the binding of all three proteins would be compatible. Collectively, these and previously published data suggest that NS1 interactions with eIF4GI and PABP1, as well as with viral mRNAs, could promote the specific recruitment of 43S complexes to the viral mRNAs.

scholarly journals Dengue Virus Utilizes a Novel Strategy for Translation Initiation When Cap-Dependent Translation Is Inhibited

2006 ◽  
Vol 80 (6) ◽  
pp. 2976-2986 ◽  
Author(s):  
Dianna Edgil ◽  
Charlotta Polacek ◽  
Eva Harris
Keyword(s):  
Dengue Virus ◽  
Translation Initiation ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Initiation Factor 4E ◽  
Cellular Translation ◽  
Cellular Mrnas ◽  
Novel Strategy

ABSTRACT Viruses have developed numerous mechanisms to usurp the host cell translation apparatus. Dengue virus (DEN) and other flaviviruses, such as West Nile and yellow fever viruses, contain a 5′ m7GpppN-capped positive-sense RNA genome with a nonpolyadenylated 3′ untranslated region (UTR) that has been presumed to undergo translation in a cap-dependent manner. However, the means by which the DEN genome is translated effectively in the presence of capped, polyadenylated cellular mRNAs is unknown. This report demonstrates that DEN replication and translation are not affected under conditions that inhibit cap-dependent translation by targeting the cap-binding protein eukaryotic initiation factor 4E, a key regulator of cellular translation. We further show that under cellular conditions in which translation factors are limiting, DEN can alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that appears not to require a functional m7G cap. This DEN noncanonical translation is not mediated by an internal ribosome entry site but requires the interaction of the DEN 5′ and 3′ UTRs for activity, suggesting a novel strategy for translation of animal viruses.

scholarly journals Functional interaction of translation initiation factor eIF4G with the foot-and-mouth disease virus internal ribosome entry site

2001 ◽  
Vol 82 (4) ◽  
pp. 757-763 ◽  
Author(s):  
Lanja Saleh ◽  
René C. Rust ◽  
Ralf Füllkrug ◽  
Ewald Beck ◽  
Gergis Bassili ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Foot And Mouth Disease ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Mouth Disease Virus ◽  
Ires Element

In the life-cycle of picornaviruses, the synthesis of the viral polyprotein is initiated cap-independently at the internal ribosome entry site (IRES) far downstream from the 5′ end of the viral plus-strand RNA. The cis-acting IRES RNA elements serve as binding sites for translation initiation factors that guide the ribosomes to an internal site of the viral RNA. In this study, we show that the eukaryotic translation initiation factor eIF4G interacts directly with the IRES of foot-and-mouth disease virus (FMDV). eIF4G binds mainly to the large Y-shaped stem–loop 4 RNA structure in the 3′ region of the FMDV IRES element, whereas stem–loop 5 contributes only slightly to eIF4G binding. Two subdomains of stem–loop 4 are absolutely essential for eIF4G binding, whereas another subdomain contributes to a lesser extent to binding of eIF4G. At the functional level, the translational activity of stem–loop 4 subdomain mutants correlates with the efficiency of binding of eIF4G in the UV cross-link assay. This indicates that the interaction of eIF4G with the IRES is crucial for the initiation of FMDV translation. A model for the interaction of initiation factors with the IRES element is discussed.

scholarly journals A Novel Form of DAP5 Protein Accumulates in Apoptotic Cells as a Result of Caspase Cleavage and Internal Ribosome Entry Site-Mediated Translation

2000 ◽  
Vol 20 (2) ◽  
pp. 496-506 ◽  
Author(s):  
Sivan Henis-Korenblit ◽  
Naomi Levy Strumpf ◽  
Dan Goldstaub ◽  
Adi Kimchi
Keyword(s):  
Internal Ribosome Entry Site ◽  
Dominant Negative ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Apoptotic Cells ◽  
Entry Site ◽  
Translation Rate ◽  
Internal Ribosome Entry ◽  
Caspase Cleavage ◽  
Induced Apoptosis

ABSTRACT Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5′ untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis.

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