Translation of most mRNAs is suppressed under stress conditions. Phosphorylation of the a-subunit of eukaryotic translation initiation factor 2 (eIF2), which delivers initiator tRNA (Met-tRNA i ) to the P site of the 40S ribosomal subunit, is responsible for such translational suppression. However, translation of hepatitis C viral (HCV) mRNA is refractory to the inhibitory effects of eIF2a phosphorylation, which prevents translation by disrupting formation of the eIF2-GTP-Met-tRNA i ternary complex. Here, we report that eIF2A, an alternative initiator tRNA-binding protein, has a key role in the translation of HCV mRNA during HCV infection, in turn promoting eIF2a phosphorylation by activating the eIF2a kinase PKR. Direct interaction of eIF2A with the IIId domain of the HCV internal ribosome entry site (IRES) is required for eIF2A-dependent translation. These data indicate that stress-independent translation of HCV mRNA occurs by recruitment of eIF2A to the HCV IRES via direct interaction with the IIId domain and subsequent loading of Met-tRNA i to the P site of the 40S ribosomal subunit.

Daily oscillations of gene expression underlie circadian behaviours in multicellular organisms1. While attention has been focused on transcriptional and posttranslational mechanisms1–3, other posttranscriptional modes have been less clearly delineated. Here we report mutants of a novel Drosophila gene twenty-four (tyf) that display weak behavioural rhythms. Weak rhythms are accompanied by dramatic reductions in the levels of the clock protein PERIOD (PER) as well as more modest effects on TIMELESS (TIM). Nonetheless, PER induction in pacemaker neurons can rescue tyf mutant rhythms. TYF associates with a 5′-cap binding complex, poly(A)-binding protein (PABP) as well as per and tim transcripts. Furthermore, TYF activates reporter expression when tethered to reporter mRNA even in vitro. Taken together, these data suggest that TYF potently activates PER translation in pacemaker neurons to sustain robust rhythms, revealing a novel and important role for translational control in the Drosophila circadian clock.

Hepatitis C virus (HCV) causes liver diseases, such as hepatitis, liver cirrhosis, steatosis, and hepatocellular carcinoma. To understand the life cycle and pathogenesis of HCV, the one-step growth of HCV in a cell culture system was analyzed using a highly infectious variant of the JFH1 clone. The observed profiles of HCV RNA replication indicated that the synthesis of negative-strand RNAs occurred at 6 h (h) after infection, followed by the active synthesis of positive-strand RNAs. Our measurements of infectious virus production showed that the latent period of HCV was about 12 h. The specific infectivity of HCV particles (focus-forming unit per viral RNA molecule) secreted to the extracellular milieu early in infection was about 30-fold higher than that secreted later during infection. The buoyant densities of the infectious virion particles differed with the duration of infection, indicating changes in the compositions of the virion particles.

Hepatitis C virus (HCV), one of the major causative agents of virus-related liver cirrhosis and hepatocellular carcinoma in humans, contains a single-stranded RNA genome of positive polarity. HCV RNA contains nontranslated regions (NTRs) at the 5Ј and 3Ј ends (18,23) and a long open reading frame encoding polyprotein that is synthesized through a single translational initiation event directed by an RNA element designated the internal ribosomal entry site (IRES) (21, 38) at the 5Ј NTR (46). The polyprotein is proteolytically processed into 10 or more viral proteins.In general, IRES elements need several canonical translation factors (except eukaryotic initiation factor 4E [eIF4E]) for their activities (40,42). However, HCV IRES-dependent translation requires only a few canonical factors (eIF2, eIF3, eIF5, and eIF5B) for function (30,39,41). Additionally, cellular proteins known as IRES-specific cellular transacting factors (ITAFs) are required for the efficient translation of HCV mRNA. For instance, polypyrimidine tract-binding protein (PTB) interacting with HCV IRES is required for IRES function (2, 3). La antigen interacting with the GCAC site near the initiator AUG is necessary for the optimal function of the HCV IRES (1, 10, 43). Recent studies have shown that NSAP1 interacting with the adenosine-rich core-coding region of HCV mRNA augments HCV IRES-dependent translation (27).Heterogeneous ribonucleoprotein D (hnRNP D), also known as AU-rich element RNA-binding protein 1 (AUF1), is an hnRNP family member that shuttles between the nucleus and cytoplasm (44, 48). hnRNP D was initially identified owing to its ability to bind and destabilize c-myc mRNA in a crude in vitro decay system (6). The protein has four isoforms of different molecular weights (p37, p40, p42, and p45), all of which are produced by alternate splicing of a single transcript (11,47). The hnRNP D protein has various functions, including mRNA decay (6), telomere maintenance (12) MATERIALS AND METHODSPlasmid construction and small interfering RNA (siRNA). Dual reporters harboring the HCV IRES, encephalomyocarditis virus (EMCV) IRES, and cmyc IRES were constructed as described previously (27). The monocistronic reporter containing the HCV IRES and EMCV IRES, followed by firefly luciferase used for in vitro translation, were prepared according to methods described in a previous report (28). Plasmids expressing hnRNP D, pFLAG-CMV2 p37, pFLAG-CMV2 p40, pFLAG-CMV2 p42, and pFLAG-CMV2 p45 were kindly provided by R. J. Schneider at New York University School of Medicine (44). To generate pRSET A-hnRNP D for recombinant hnRNP D purification, pFLAG-CMV2 p45 was treated with HindIII-Klenow-EcoRI, and the resulting DNA fragment was cloned into NcoI-Klenow-EcoRI-treated pRSET A (Invitrogen).To construct pH(130-228)CAT and pH(229-402)CAT used for generating an RNA probe (see Fig. 2B, below), HCV IRES corresponding to positions 130 to 228 and 229 to 402 were amplified from pH(18-402)CAT (27) using the following primer pairs: 5Ј-CTAGGTACCGGGAGAGCCATAG-3Ј and 5...

Frameshift and nonsense mutations are common in tumors with microsatellite instability, and mRNAs from these mutated genes have premature termination codons (PTCs). Abnormal mRNAs containing PTCs are normally degraded by the nonsense-mediated mRNA decay (NMD) system. However, PTCs located within 50–55 nucleotides of the last exon–exon junction are not recognized by NMD (NMD-irrelevant), and some PTC-containing mRNAs can escape from the NMD system (NMD-escape). We investigated protein expression from NMD-irrelevant and NMD-escape PTC-containing mRNAs by Western blotting and transfection assays. We demonstrated that transfection of NMD-irrelevant PTC-containing genomic DNA of MARCKS generates truncated protein. In contrast, NMD-escape PTC-containing versions of hMSH3 and TGFBR2 generate normal levels of mRNA, but do not generate detectable levels of protein. Transfection of NMD-escape mutant TGFBR2 genomic DNA failed to generate expression of truncated proteins, whereas transfection of wild-type TGFBR2 genomic DNA or mutant PTC-containing TGFBR2 cDNA generated expression of wild-type protein and truncated protein, respectively. Our findings suggest a novel mechanism of gene expression regulation for PTC-containing mRNAs in which the deleterious transcripts are regulated either by NMD or translational repression.

Eukaryotic translation initiation commences at the initiation codon near the 5′ end of mRNA by a 40S ribosomal subunit, and the recruitment of a 40S ribosome to an mRNA is facilitated by translation initiation factors interacting with the m 7 G cap and/or poly (A) tail. The 40S ribosome recruited to an mRNA is then transferred to the AUG initiation codon with the help of translation initiation factors. To understand the mechanism by which the ribosome finds an initiation codon, we investigated the role of eIF4G in finding the translational initiation codon. An artificial polypeptide eIF4G fused with MS2 was localized downstream of the reporter gene through MS2-binding sites inserted in the 3′ UTR of the mRNA. Translation of the reporter was greatly enhanced by the eIF4G-MS2 fusion protein regardless of the presence of a cap structure. Moreover, eIF4G-MS2 tethered at the 3′ UTR enhanced translation of the second cistron of a dicistronic mRNA. The encephalomyocarditis virus internal ribosome entry site, a natural translational-enhancing element facilitating translation through an interaction with eIF4G, positioned downstream of a reporter gene, also enhanced translation of the upstream gene in a capindependent manner. Finally, we mathematically modeled the effect of distance between the cap structure and initiation codon on the translation efficiency of mRNAs. The most plausible explanation for translational enhancement by the translational-enhancing sites is recognition of the initiation codon by the ribosome bound to the ribosome-recruiting sites through "RNA looping." The RNA looping hypothesis provides a logical explanation for augmentation of translation by enhancing elements located upstream and/or downstream of a protein-coding region.RNA looping | translation initiation | ribosome scanning | eukaryotic mRNA T ranslation initiation is complex process in which more than 10 kinds of proteins participate (1). During the first event of translation initiation, it is believed that the 43S preinitiation complex, composed of 40S ribosome, eIF3, eIF5, eIF1, eIF1A, and ternary complex (eIF2-GTP initiator tRNA), is recruited to a 5′ cap structure at the end of mRNA via a preexisting mRNAeIF4F (eIF4E, eIF4A, and eIF4G) complex through a proteinprotein interaction between eIF4G and eIF3 (1). The resulting 40S ribosomal subunit-containing complex, called the 43S preinitiation complex, moves to the initiation codon. Although most of the eukaryotic mRNAs use the cap structure at the 5′ end when recruiting the 40S ribosome, some mRNAs use a specialized RNA element, the internal ribosome entry site (IRES), for recruiting the 40S ribosome to mRNA (2, 3). eIF4G protein plays a pivotal role in both cap-dependent and IRES-dependent translations, not only for ribosome recruitment, but also for initiation codon selection. eIF4G is a scaffold protein that links the 43S ribosomal complex and mRNA. In the cap-dependent translation, eIF4G is loaded onto mRNA as a protein complex with eIF4E (cap-binding protein) and eIF4A (RNA helicase...

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Translation of many cellular and viral mRNAs is directed by internal ribosomal entry sites (IRESs). Several proteins that enhance IRES activity through interactions with IRES elements have been discovered. However, the molecular basis for the IRES-activating function of the IRES-binding proteins remains unknown. Here, we report that NS1-associated protein 1 (NSAP1), which augments several cellular and viral IRES activities, enhances hepatitis C viral (HCV) IRES function by facilitating the formation of translation-competent 48S ribosome–mRNA complex. NSAP1, which is associated with the solvent side of the 40S ribosomal subunit, enhances 80S complex formation through correct positioning of HCV mRNA on the 40S ribosomal subunit. NSAP1 seems to accomplish this positioning function by directly binding to both a specific site in the mRNA downstream of the initiation codon and a 40S ribosomal protein (or proteins).