John W. B. Hershey

EFFECT OF INITIATION FACTOR eIF-5A DEPLETION ON CELL PROLIFERATION AND PROTEIN SYNTHESIS

Initiation factor eIF-5A (formerly called eIF-4D) was originally isolated (Kemper et al., 1976; Benne et al., 1978) from rabbit reticulocytes based on its activity in stimulating the synthesis of methionyl-puromycin, a model of the formation of the first peptide bond in protein synthesis. This highly conserved protein is small (ca. 18 kDa) and acidic (pI = 5.4) and is one of the most abundant initiation factors in mammalian cells. eIF-5A is unique in undergoing a covalent post-translational modification by a two-step pathway that involves the attachment of an aminobutyl group from spermidine to the e-amino group of a lysine residue, followed by hydroxylation on the number 2 carbon of the butyl group, to form a hypusine residue (Park et al., 1984). The hypusination reaction correlates with stimulation of cell growth and protein synthesis, thereby eliciting considerable interest in the protein.

The Initiation Factors

Initiation of protein synthesis is the process whereby the ribosome binds mRNA and the first aminoacyl-tRNA to form an initiation complex which is capable of entering the elongation phase of protein synthesis. The pathway is complex and involves numerous steps: First, the 80S ribosome dissociates into 40S and 60S ribosomal subunits; the 40S subunit forms a preinitiation complex with methionyl-tRNA and mRNA; this is joined by the 60S subunit to complete formation of the 80S initiation complex. During these steps, two critical events occur: the ribosome selects for translation a specific mRNA from among numerous species of mRNAs; and the methionyl-tRNA interacts with a specific initiator site on the mRNA to assure proper translation in the correct phase. The reactions are promoted or catalyzed by a complex array of initiation factors and involve the hydrolysis of ATP and GTP.

Mapping surface residues of eIF5A that are important for binding to the ribosome using alanine scanning mutagenesis

The translation elongation factor eIF5A is conserved through evolution and is necessary to rescue the ribosome during translation elongation of polyproline-containing proteins. Although the site of eIF5A binding to the ribosome is known, no systematic analysis has been performed so far to determine the important residues on the surface of eIF5A required for ribosome binding. In this study, we used clustered charged-to-alanine mutagenesis and structural modeling to address this question. We generated four new mutants of yeast eIF5A: tif51A-4, tif51A-6, tif51A-7 and tif51A-11, and complementation analysis revealed that tif51A-4 and tif51A-7 could not sustain cell growth in a strain lacking wild-type eIF5A. Moreover, the allele tif51A-4 also displayed negative dominance over wild-type eIF5A. Both in vivo GST-pulldowns and in vitro fluorescence anisotropy demonstrated that eIF5A from mutant tif51A-7 exhibited an importantly reduced affinity for the ribosome, implicating the charged residues in cluster 7 as determinant features on the eIF5A surface for contacting the ribosome. Notably, modified eIF5A from mutant tif51A-4, despite exhibiting the most severe growth phenotype, did not abolish ribosome interactions as with mutant tif51A-7. Taking into account the modeling eIF5Axa0+xa080Sxa0+xa0P-tRNA complex, our data suggest that interactions of eIF5A with ribosomal protein L1 are more important to stabilize the interaction with the ribosome as a whole than the contacts with P-tRNA. Finally, the ability of eIF5A from tif51A-4 to bind to the ribosome while potentially blocking physical interaction with P-tRNA could explain its dominant negative phenotype.

Structure/Function of Mammalian Initiation Factors

The initiation phase of protein synthesis in eukaryotic cells is promoted by at least 10 different initiation factor proteins, comprising over 25 distinct polypeptides. The complexity of the process and the finding that these proteins are post-translationally modified by phosphorylation inter al., suggest that rates of translation are controlled by regulating initiation factor activities. In order to better understand the functional roles played by the initiation factors, we and others have purified the proteins from mammalian cells and have begun to elucidate their structures by cloning cDNAs encoding the factors. Possession of cDNA clones provides not only the protein’s sequence, but also tools for cloning the gene and studying its expression, for overexpressing the protein in cells, and for manipulating the protein’s structure. This allows us to study the function of the initiation factors in vivo, thereby providing confirmation of results obtained by in vitro biochemical experiments.

RNA Helicases and Their Cofactors

RNA helicases are proteins that function by melting RNA secondary structures or remodeling ribonucleoprotein complexes. Canonical RNA helicase eIF4A plays an essential role in translation initiation by resolving secondary structures in the 5′ UTR of mRNAs and preparing the mRNA template for ribosome recruitment. Interestingly, the majority of mRNAs that encode proteins responsible for cell survival, proliferation, cell cycle transitions and angiogenesis contain 5′ UTRs with significant secondary/tertiary structures, and thus, appear to be more dependent on the RNA helicase activity of eIF4A. In addition, several other RNA helicases have been implicated in other steps of translation initiation, as well as in exerting mRNA-specific regulation. In this chapter, we discuss the RNA helicases implicated in translational control, as well as their role in cancer biology and their potential as diagnostic, prognostic and therapeutic targets.

Translational Control by Phosphorylation of Mammalian Initiation Factors

Translational control is defined as a change in the efficiency of translation of mRNAs. This may involve a quantitative change in the overall amount of protein synthesized, called global control. Alternatively, the change may affect the synthesis of specific proteins, called mRNA-specific control. In either case, efficiency of translation is usually measured by radiolabeled amino acid incorporation into protein, or by the number and activity of ribosomes. Since the rate limiting step in protein synthesis appears to be at the initiation phase, most control mechanisms are thought to regulate this step. We therefore focus on the pathway of initiation and address studies of how global rates of protein synthesis may be controlled.

The Structure and Regulation of Mammalian Initiation Factor eIF2

The process of protein synthesis on ribosomes is promoted by soluble protein factors that act during the initiation, elongation and termination phases of translation (for reviews, see Moldave, 1985; Pain, 1986). A striking characteristic of these factors is that most of them bind GTP or GDP. Since the structure and mechanism of action of these proteins have been studied extensively for the past 20 years, considerable knowledge of their mechanisms of action has accummulated which may be relevant to understanding how G-proteins function in general. We are concerned here with one of the mammalian factors, initiation factor eIF2.

Messenger Ribonucleoprotein Particles

Messenger RNA, from the time of its synthesis in the nucleus through its translation and degradation, does not exist as free RNA in animal cells but rather always appear to be complexed with proteins. Free cytoplasmic ribonucleoprotein (RNP) particles containing non-ribosomal RNA were discovered by Spirin and coworkers (1) in 1964 and were called informosomes. Shortly thereafter, similar particles containing hnRNA were identified in cell nuclei (2). Both kinds of mRNP particles are heterogeneous in size and are rich in protein, with RNA/protein mass ratios of about 1:3. mRNPs have been found in all eukaryotic organisms examined.