The 5' ends of eukaryotic mRNAs are blocked by a cap structure, m7GpppX (where X is any nucleotide). The interaction of the cap structure with a cap-binding protein complex is required for efficient ribosome binding to the mRNA. In Saccharomyces cerevisiae, the cap-binding protein complex is a heterodimer composed of two subunits with molecular masses of 24 (eIF-4E, CDC33) and 150 (p150) kDa. p150 is presumed to be the yeast homolog of the p220 component of mammalian eIF-4F. In this report, we describe the isolation of yeast gene TIF4631, which encodes p150, and a closely related gene, TIF4632. TIF4631 and TIF4632 are 53% identical overall and 801% identical over a 320-amino-acid stretch in their carboxy-terminal halves. Both proteins contain sequences resembling the RNA recognition motif and auxiliary domains that are characteristic of a large family of RNA-binding proteins. tif4631-disrupted strains exhibited a slow-growth, cold-sensitive phenotype, while disruption of TIF4632 failed to show any phenotype under the conditions assayed. Double gene disruption engendered lethality, suggesting that the two genes are functionally homologous and demonstrating that at least one of them is essential for viability. These data are consistent with a critical role for the high-molecular-weight subunit of putative yeast eIF-4F in translation. Sequence comparison of TIF4631, TIF4632, and the human eIF-4F p220 subunit revealed significant stretches of homology. We have thus cloned two yeast homologs of mammalian p220.The 5'-terminal cap structure m7GpppX (where X is any nucleotide) is required for efficient mRNA translation and plays a prominent role in translational control. This ubiquitous feature of eukaryotic mRNAs is also important for nuclear events. Precursor mRNA splicing (23, 46) and 3'-end processing (26, 34) are enhanced by the presence of a cap structure. In addition, the cap structure protects the mRNA against 5' exonucleolytic degradation in both the nucleus and the cytoplasm (25,30) and is implicated in nucleocytoplasmic transport (33). The best-characterized role of the cap structure is its stimulatory effect on ribosome binding (for reviews, see references 66 and 82).

The 5' cap structure m7GpppN (where N is any nucleotide) is a ubiquitous feature of cellular eukaryotic mRNAs. The cap is multifunctional as it is involved in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against 5' exonucleolytic degradation. The cap binding protein, eukaryotic initiation factor 4E (eIF-4E), is a translation initiation factor that binds to the cap structure and is part of a complex (eIF-4F) that promotes mRNA binding to ribosomes. Overexpression of eIF-4E in fibroblasts results in cell. transformation. To test the hypothesis that some of the biological effects of eIF-4E might be effected by a nuclear function, we determined the cellular distribution of eIF-4E. By means of indirect immunofluorescence experiments using polyclonal and monoclonal antibodies against eIF-4E as well as transfected epitope-tagged eIF-4E, we demonstrate that a fraction ofeIF-4E localizes to the nucleus. These results suggest that eIF-4E is also involved in a nuclear function.The cap structure of eukaryotic mRNAs is added early during the transcription of RNA polymerase II genes in the nucleus (1) and plays an important role in several cytoplasmic and nuclear biochemical processes: (i) translational initiation (2), (ii) precursor mRNA splicing (3, 4), (iii) mRNA 3'-end processing (5, 6), (iv) mRNA nucleocytoplasmic transport (7), and (v) protection of mRNA against 5' exonucleolytic degradation in the cytoplasm and nucleus (8). Several proteins that recognize and bind the cap have been identified in the cytoplasm and the nucleus. These proteins are likely to mediate the disparate functions of the cap. Indeed, a large body of evidence shows that a polypeptide of 24 kDa, termed eukaryotic initiation factor 4E (eIF-4E), mediates cap function during translation initiation (9, 10). However, relatively little is known about the proteins that function in capdependent nuclear processes. Several nuclear cap-binding proteins have been identified by UV-induced cross-linking to the cap structure. These include polypeptides of 20 and 115 kDa (11) as well as 80, 89, and 120 kDa (12). More recently, an 80-kDa cap-binding protein has been purified from HeLa nuclear extracts (13). The function(s) of the nuclear capbinding proteins is not known.eIF-4E was initially identified and purified from the highsalt wash of ribosomes prepared from rabbit reticulocyte lysate (14, 15) and subsequently was detected in the postribosomal supernatant (16). It has been purified from different species including plants (17,18), yeast (19), and Drosophila (20). eIF4E can be purified as a single polypeptide or as part of a complex (termed eIF4F) with one or two other polypeptides. In all species, a subunit of 150-220 kDa is associated with eIF-4E in the complex (17,(21)(22)(23)(24) (30). In addition, eIF-4E cooperates with nuclear oncogenes such as c-myc or EJA to transform primary cells (31). The mechanism by which eIF-4E transforms cells in culture is not clear. To test the hypothesis that eIF-4E might have nuclear fu...

Nucleotide cap analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized in which the 7-methyl moiety was replaced with 7-ethyl (e7), 7-propyl (p7), 7-isopropyl (ip7), 7-butyl (b7), 7-isobutyl (ib7), 7-cyclopentyl (cp7), 7-(carboxymethyl) (cm7), 7-benzyl (bn7), 7-(2-phenylethyl) [7-(2-PhEt)], and 7-(1-phenylethyl) [7-(1-PhEt)]. These derivatives were assayed as competitive inhibitors of capped mRNA translation in reticulocyte lysate. We observed that N7 alkyl and alicyclic substituents larger than ethyl significantly decreased the inhibitory activity of these cap analogues presumably by decreasing their affinity for cap binding proteins, which participate in the initiation of translation. This result defined a maximum size for this class of N7 substituents in the nucleotide binding domain of cap binding proteins. Like m7GMP, the N7-substituted GMP derivatives synthesized in this study were found to be predominantly in the anti conformation as determined by proton NMR analyses. However, bn7GMP and 7-(2-PhEt)GMP, which have aromatic N7 substituents, were more effective than m7GMP as competitive inhibitors of translation. The increased affinity of bn7GMP for cap binding proteins was further examined by synthesis of beta-globin mRNA containing 5'-bn7G, 5'-m7G, or 5'-e7G cap structures. These modified mRNAs were tested as translation templates. Messenger RNA capped with bn7G was observed to increase the translation activity of the template 1.8-fold relative to that of its m7G-capped mRNA counterpart. By contrast, e7G-capped mRNA was 25% less active than m7G-capped mRNA.2+V photo-cross-linking of m7G-capped mRNA to cap binding proteins

The eukaryotic mRNA 5' cap structure m7GpppX (where X is any nucleotide) interacts with a number of cellular proteins. Several of these proteins were studied in mammalian, yeast, and drosophila cells and found to be involved in translation initiation. Here we describe a novel cap-binding protein, the coat protein of L-A, a double-stranded RNA virus that is persistently maintained in many Saccharomyces cerevisiae strains. The results also suggest that the coat protein of a related double-stranded RNA virus (L-BC) is likewise a cap-binding protein. Strikingly, in contrast to the cellular cap-binding proteins, the interaction between the L-A virus coat protein and the cap structure is through a covalent bond.All eukaryotic cellular mRNAs (except for organellar mRNAs) possess the 5' structure m7GpppX (where X is any nucleotide), termed cap. The cap structure plays important roles in several cytoplasmic and nuclear processes.

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