The mechanism of ribosome binding to eucaryotic mRNAs is not well understood, but it requires the participation of eucaryotic initiation factors eIF-4A, eIF-4B, and eIF-4F and the hydrolysis of ATP. Evidence has accumulated in support of a model in which these initiation factors function to unwind the 5'-proximal secondary structure in mRNA to facilitate ribosome binding. To obtain direct evidence for initiation factor-mediated RNA unwinding, we developed a simple assay to determine RNA helicase activity, and we show that eIF-4A or eIF-4F, in combination with eIF-4B, exhibits helicase activity. A striking and unprecedented feature of this activity is that it functions in a bidirectional manner. Thus, unwinding can occur either in the 5'-to-3' or 3'-to-5' direction. Unwinding in the 5'-to-3' direction by eIF-4F (the cap-binding protein complex), in conjunction with eIF-4B, was stimulated by the presence of the RNA 5' cap structure, whereas unwinding in the 3'-to-5' direction was completely cap independent. These results are discussed with respect to cap-dependent versus cap-independent mechanisms of ribosome binding to eucaryotic mRNAs.A critical step in eucaryotic protein biosynthesis is binding of the small (40S) ribosomal subunit to mRNA (36,39). This step is rate limiting in translation initiation (25) and is a key target for regulation (reviewed in reference 53), but the mechanism of this process is poorly understood. Two pathways for the binding of 40S ribosomal subunits to mRNA have been described, which differ in their requirement for the cap structure. The 5' cap structure, m7GpppX (where X is any nucleotide) is a nearly ubiquitous feature of all eucaryotic mRNAs (49). Evidence indicates that translation initiation of the majority of eucaryotic mRNAs is accomplished in a cap-enhanced manner, whereby 40S ribosomal binding to mRNA is facilitated by the cap structure (reviewed in references 3 and 50). Recently, it has been shown that poliovirus (42) and encephalomyocarditis virus (26) mRNAs, which are naturally uncapped (13,21,38), initiate translation by a different mechanism. In this case, the 40S subunit binds directly to an internal element on the picornavirus 5' untranslated region, by-passing upstream sequences and the requirement for the cap structure.Although the two initiation pathways are mechanistically distinguishable, they nevertheless require a similar set of initiation factors (eIFs [57]) to bind 40S ribosomal subunits to mRNA. Cap-stimulated mRNA binding to the small ribosomal subunit requires at least three initiation factors, eIF-4A, eIF-4B, and eIF-4F, in addition to the hydrolysis of ATP (reviewed in references 10, 44, and 53). eIF-4F is a multisubunit complex consisting of three major polypeptides of 24, 50, and 220 kilodaltons (kDa) (8,20,59). The 24-kDa polypeptide is the cap-binding subunit, which also exists in a free form, termed eIF-4E (55). The 50-kDa polypeptide is a structural variant of free eIF-4A (8,20). Although eIF-4F contains an eIF-4A subunit which is almost identical to...

Translation initiation on poliovirus RNA occurs by internal binding of ribosomes to a sequence within the 5' untranslated region. We have previously characterized a HeLa cell protein, p52, that binds to a fragment of the poliovirus 5' untranslated region (K. Meerovitch, J. Pelletier, and N. Sonenberg, Genes Dev. 3:1026-1034, 1989). Here we report the purification of the HeLa p52. Protein microsequencing identified p52 as La autoantigen. The La protein is a human antigen that is recognized by antibodies from patients with autoimmune disorders such as systemic lupus erythematosus and Sjogren's syndrome. We show that the La protein stimulates translation of poliovirus RNA, but not brome mosaic virus, tobacco mosaic virus, and alfalfa mosaic virus 4 RNA, translation in a reticulocyte lysate. In addition, La corrects aberrant translation of poliovirus RNA in a reticulocyte lysate. Subcellular immunolocalization showed that La protein is mainly nuclear, but after poliovirus infection, La is redistributed to the cytoplasm. Our results suggest that La protein is involved in poliovirus internal initiation of translation and might function through a similar mechanism in the translation of cellular mRNAs.

Initiation of translation on poliovirus mRNA occurs by internal binding of ribosomes to a region within the 5'-noncoding portion of the mRNA. The mechanistic details and trans-acting factors involved in this event are not understood fully. We used a mobility-shift electrophoresis assay to identify a specific RNA-protein complex, which can form between an RNA fragment that contains nucleotides 559--624 of the poliovirus 5' UTR {untranslated region) and a component or components of a HeLa cell extract. Complex formation was reduced greatly in a reticulocyte lysate or a wheat-germ extract. A 52-kD polypeptide (p52) has been identified as part of the protein-RNA complex by use of an UV cross-linking assay. This polypeptide apparently is not a known translation initiation or elongation factor. The possible involvement of p52 in translation initiation of poliovirus protein synthesis is discussed.

By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (1a) has been identified as a potent apoptosis inducer. Compound 1a was found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G(2)/M stage and to induce apoptosis as determined by the flow cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). Through structure-activity relationship (SAR) studies of the 4-aryl group, a 4- and 7-fold increase in potency was obtained from the screening hit 1a to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethylamino)-4H-chromene (1c) and 2-amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-pyridyl)-4H-chromene (4e), with an EC(50) of 19 and 11 nM in the caspase activation assay in T47D breast cancer cells, respectively. The 2-amino-4-aryl-3-cyano-7-(dimethylamino)-4H-chromenes also were found to be highly active in the growth inhibition MTT assay, with GI(50) values in the low nanomolar range for compound 1c. Significantly, compound 1c was found to have a GI(50) value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MES-SA/DX5 tumor cells. Functionally, compound 1c was found to be a potent inhibitor of tubulin polymerization and to effectively inhibit the binding of colchicine to tubulin. These results confirm that the cell-based caspase activation assay is a powerful tool for the discovery of potent apoptosis inducers and suggest that the 4-aryl-4H-chromenes have the potential to be developed into future anticancer agents.

Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of transacting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNAprogrammed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.