La Autoantigen Is Necessary for Optimal Function of the Poliovirus and Hepatitis C Virus Internal Ribosome Entry Site In Vivo and In Vitro

Department of Biochemistry and McGill Cancer Center, McGill University, McIntyre Medical Building, Montreal, Quebec, Canada H3G 1Y6.
Molecular and Cellular Biology (Impact Factor: 4.78). 09/2004; 24(15):6861-70. DOI: 10.1128/MCB.24.15.6861-6870.2004
Source: PubMed


Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5' untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

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Available from: Yuri Svitkin, Jun 25, 2014
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    • "Analysis of the 48S preinitiation complex formation was performed as described (11,24). Briefly, VEGF-IRES plasmids was linearized, in vitro transcribed and radiolabeled using a MAXIscript SP6 kit (Ambion) in the presence of 50 μCi of [α-32P]UTP (Perkin-Elmer). "
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    ABSTRACT: Dyskerin is a nucleolar protein encoded by the DKC1 gene that (i) stabilizes the RNA component of the telomerase complex, and (ii) drives the site-specific pseudouridilation of rRNA. It is known that the partial lack of dyskerin function causes a defect in the translation of a subgroup of mRNAs containing internal ribosome entry site (IRES) elements such as those encoding for the tumor suppressors p27 and p53. In this study, we aimed to analyze what is the effect of the lack of dyskerin on the IRES-mediated translation of mRNAs encoding for vascular endothelial growth factor (VEGF). We transiently reduced dyskerin expression and measured the levels of the IRES-mediated translation of the mRNA encoding for VEGF in vitro in transformed and primary cells. We demonstrated a significant increase in the VEGF IRES-mediated translation after dyskerin knock-down. This translational modulation induces an increase in VEGF production in the absence of a significant upregulation in VEGF mRNA levels. The analysis of a list of viral and cellular IRESs indicated that dyskerin depletion can differentially affect IRES-mediated translation. These results indicate for the first time that dyskerin inhibition can upregulate the IRES translation initiation of specific mRNAs.
    Nucleic Acids Research 07/2013; 41(17). DOI:10.1093/nar/gkt587 · 9.11 Impact Factor
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    • "Type I and II IRESs also utilize non-canonical translation initiation factors, termed IRES transacting factors (ITAFs). Examples of ITAFs include La autoantigen, pyrimidine tract binding (PTB) protein, and upstream of N-Ras (UNR; Costa-Mattioli et al., 2004; Cornelis et al., 2005; Verma et al., 2010). ITAFs allow for the bypass of canonical translation initiation factors that are likely the target of viral strategy, either through direct proteolytic cleavage or modulation of pathways (such as UPR modulation) that negate their function. "
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    ABSTRACT: Many virus infections and stresses can induce endoplasmic reticulum (ER) stress response, a host self-defense mechanism against viral invasion and stress. During this event, viral and cellular gene expression is actively regulated and often encounters a switching of the translation initiation from cap-dependent to internal ribosome-entry sites (IRES)-dependent. This switching is largely dependent on the mRNA structure of the 5' untranslated region (5' UTR) and on the particular stress stimuli. Picornaviruses and some other viruses contain IRESs within their 5' UTR of viral genome and employ an IRES-driven mechanism for translation initiation. Recently, a growing number of cellular genes involved in growth control, cell cycle progression and apoptosis were also found to contain one or more IRES within their long highly structured 5' UTRs. These genes initiate translation usually by a cap-dependent mechanism under normal physiological conditions; however, in certain environments, such as infection, starvation, and heat shock they shift translation initiation to an IRES-dependent modality. Although the molecular mechanism is not entirely understood, a number of studies have revealed that several cellular biochemical processes are responsible for the switching of translation initiation to IRES-dependent. These include the cleavage of translation initiation factors by viral and/or host proteases, phosphorylation (inactivation) of host factors for translation initiation, overproduction of homologous proteins of cap-binding protein eukaryotic initiation factors (eIF)4E, suppression of cap-binding protein eIF4E expression by specific microRNA, activation of enzymes for mRNA decapping, as well as others. Here, we summarize the recent advances in our understanding of the molecular mechanisms for the switching of translation initiation, particularly for the proteins involved in cell survival and apoptosis in the ER stress pathways during viral infections.
    Frontiers in Microbiology 03/2012; 3:92. DOI:10.3389/fmicb.2012.00092 · 3.99 Impact Factor
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    • "The versatility of La in its association with RNA (49) suggests that it may bind to multiple sites in the PV IRES. Even though La was demonstrated to bind to the stem–loop VI of the PV IRES (19), no systematic mapping of possible other La binding sites has been reported, and the molecular action of La in the PV translation is not yet understood (50). "
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    ABSTRACT: Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5' end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNA(Gly) anticodon stem-loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus-host cell interaction are discussed.
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