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: 5.04). 09/2004; 24(15):6861-70. DOI: 10.1128/MCB.24.15.6861-6870.2004
Source: PubMed

ABSTRACT 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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    • "Since the interaction of the La protein is weaker with mutant RNA, we have investigated the IRES activities of the wt and mutant RNA in cells that are partially silenced of La gene expression. Co-transfection of plasmid DNA corresponding to the wt or mutant CVB3 59 UTR bicistronic construct was performed in HeLa cells, with precharacterized siRNA targeting endogenous La mRNA (Costa-Mattioli et al., 2004). A non-specific siRNA was used as negative control. "
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    • "Many cellular proteins interact with the picornavirus 59 UTR and regulate virus replication; for instance, poly(rC)binding protein (PCBP), an RNA-binding protein that contains three heterogeneous nuclear ribonucleoprotein K (hnRNP K) homology (KH) motifs, has been demonstrated to interact with the 59 UTR of poliovirus and rhinovirus (Walter et al., 1999, 2002). Various IRESspecific trans-acting factors, such as polypyrimidine tractbinding protein (PTB), PCBP, autoantigen La and upstream N-ras protein (Unr), have been reported to be functionally important for picornaviruses (Boussadia et al., 2003; Costa-Mattioli et al., 2004; Toyoda et al., 1994; Walter et al., 1999). "
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    • "Conformational changes are thought to occur during factor assembly on the IRES⅐40S complex (Spahn et al., 2001; Boehringer et al., 2005) and could facilitate this de novo assembly process. The recruitment of eIF2 or Met-tRNA i Met to HCV-bound 40S ribosomes could be facilitated by other auxiliary factors such as La autoantigen (Ali and Siddiqui, 1997; Costa-Mattioli et al., 2004) or polypyrimidine tract-binding protein (Anwar et al., 2000). It is worthwhile to note that the yeast homologue of La, LHP1, can act as an RNA chaperone to stabilize specific conformations of tRNA (Yoo and Wolin, 1997), and a similar protein could help recruit the Met-tRNA i Met to the IRES⅐40S complex. "
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