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Eukaryotic initiation factor (eIF) 4B is part of the protein complex involved in the recognition and binding of mRNA to the ribosome. DrosophilaeIF4B is a single-copy gene that encodes two isoforms, termed eIF4B-L (52.2 kDa) and eIF4B-S (44.2 kDa), generated as a result of the alternative recognition of two polyadeynlation signals during transcription termination and subsequent alternative splicing of the two pre-mRNAs. Both eIF4B mRNAs and proteins are expressed during the entire embryogenesis and life cycle. The proteins are cytoplasmic with polarized distribution. The two isoforms bind RNA with the same affinity. eIF4B-L and eIF4B-S preferentially enhance cap-dependent over IRES-dependent translation initiation in a Drosophila cell-free translation system. RNA interference experiments suggest that eIF4B is required for cell survival, although only a modest reduction in rate of protein synthesis is observed. Overexpression of eIF4B in Drosophila cells in culture and in developing eye imaginal discs promotes cell proliferation.

All organisms from bacteria to man respond to an exposure to higher than physiological temperatures by reprogramming their gene expression, leading to the increased synthesis of a unique set of proteins termed heat shock proteins (hsps). The hsps function as molecular chaperones in both normal and stressed cells. The rapid and efficient synthesis of hsps is achieved as a result of changes occurring at gene transcription, RNA processing and degradation, and mRNA translation. With regard to the translational regulation, the emerging picture is that the two key steps of polypeptide chain initiation, namely mRNA binding and Met-tRNA(i) binding to ribosomes, are regulated in heat-shocked mammalian cells. In Drosophila, mRNA binding is regulated by a structural feature of the leader of heat shock mRNAs and by the inactivation of eukaryotic initiation factor- (eIF-) 4F. No clear evidence for changes in Met-tRNA(i) binding has been obtained yet.

The mechanism for guanine nucleotide exchange with eukaryotic initiation factor-2 (eIF-2) from Drosophila melanogaster embryos was studied using the reaction eIF-2 X [3H]GDP + GDP (GTP) in equilibrium eIF-2 X GDP (GTP) + [3H]GDP. When highly purified eIF-2 is used the rate of nucleotide exchange is greatly reduced by Mg2+ and this reduction is overcome by the guanine-nucleotide-exchange factor (GEF) of rabbit reticulocytes. This GEF-dependent exchange is inhibited when Drosophila eIF-2 is either phosphorylated by the hemin-controlled inhibitor (HCI) of rabbit reticulocytes or treated with phosphatidylserine or a rabbit eIF-2 X phosphatidylserine complex. The Mg2+ impairment of guanine nucleotide exchange is less severe when highly purified eIF-2 is incubated at a higher temperature (37 degrees C) and is not observed at any temperature if partially purified eIF-2 is used instead of the highly purified factor. In the latter two cases the exchange is not inhibited by either phosphorylation with HCI or phospholipid treatment of Drosophila eIF-2, possibly suggesting that the observed exchange is not mediated by a GEF-like factor. Our data support two possible mechanisms for GDP/GTP exchange with Drosophila embryos eIF-2: a GEF-dependent exchange, similar to that described in rabbit reticulocytes, which may be regulated by phosphorylation of eIF-2, and a factor-independent exchange which appears to be insensitive to this type of control.

Cell-free preparations from encapsulated Artemia salina embryos readily translate poly(U), but there is no endogenous protein synthesis or translation of added natural mRNA until development resumes upon incubation in saline. High-salt-washed 80S ribosomes and highspeed supernatant (cytosol) were prepared from undeveloped eggs and from eggs allowed to develop to a stage prior to hatching. Endogenous protein synthesis occurs only with ribosomes from developed embryos, whether with undeveloped or developed cytosol. This is mainly elongation of preformed polypeptide chains, for it is largely resistant to edeine, an inhibitor of chain initiation. Edeinesensitive translation of added natural mRNA occurs with both developed and undeveloped ribosomes but requires developed cytosol. Translation of brome mosaic virus RNA is not much, if at all, further stimulated by high-saltwash of developed ribosomes, but that of globin mRNA is markedly enhanced. Levels of the chain initiation factor EIF-1 are the same before and after development. These results are consistent with the view that resumption of developemtn is triggered by transcription, with ensuing translation of the resulting messengers to yield, among other proteins, mRNA-recognizing initiation factors of the IF-3 type which are partly free in the cytosol and partly ribosome-bound. The data also suggest that different factors may be involved in the translation of brome mosaic virus RNA and globin mRNA by this system.

The initiation inhibitor of reticulocyte lysates has been shown by others to be associated with a 3':5'-cyclic-AMP-independent protein kinase that catalyzes the phosphorylation of the small (38,000 daltons) subunit of the polypeptide chain initiation factor eIF-2. This factor forms a ternary complex with Met-tRNAi and GTP which, on interaction with a 40S ribosome, gives rise to a 40S complex. Ternary complex formation is inhibited by prior incubation of partially purified eIF-2 with reticulocyte inhibitor and ATP. The relation between phosphorylation and inactivation of eIF-2 is indicated by the lack of inhibition when ATP is omitted. Translation in hemin-containing reticulocyte lysates is also inhibited by cyclic-AMP-dependent protein kinases or their catalytic subunits. They act by converting proinhibitor (inactive eIF-2 kinase) present in lysates to inhibitor (active eIF-2 kinase). This reaction is analogous to the conversion of inactive phosphorylase kinase to active phosphorylase kinase.

The formation of translational inhibitor (active eIF-2 kinase) from proinhibitor (inactive eIF-2 kinase) in reticulocyte lysates, known to be controlled by hemin, can, as we recently reported, be induced by 3':5'-cyclic AMP(cAMP)-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) or its catalytic subunit. We find that in crude preparations from rabbit reticulocyte lysates, hemin inhibits the conversion of proinhibitor to inhibitor catalyzed by endogenous cAMP-dependent protein kinase upon addition of cAMP, but not that caused by the addition of free protein kinase catalytic subunit. Hemin prevents the binding of cAMP to the regulatory subunit of cAMP-dependent protein kinase and blocks the cAMP-induced dissociation of regulatory and catalytic subunits of the enzyme whereby the enzyme is inactivated. The mechanism by which hemin prevents the formation of the inhibitor and maintains protein synthesis in reticulocyte lysates is thus explained.

The catalytic subunit of cyclic 3':5'-AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) inhibits translation in Artemia salina and wheat germ extracts. It acts, as in reticulocyte lysates [Datta, A., de Haro, C., Sierra, J. M. & Ochoa, S. (1977) Proc. Natl. Acad. Sci. USA 74, 1463-1467] by catalyzing the conversion of a proinhibitor to an inhibitor of polypeptide chain initiation. Addition of ATP and either cyclic AMP or catalytic subunit promotes the proinhibitor-inhibitor conversion in crude proinhibitor preparations from A. salina embryos. The effect of cyclic AMP is due to stimulation of cyclic AMP-dependent protein kinase, present in such preparations, and is inhibited by hemin. In similar preparations from wheat germ, addition of ATP and catalytic subunit promoted proinhibitor-inhibitor conversion, but addition of ATP and cyclic AMP has little or no effect. As assayed with histone as substrate, wheat germ preparations exhibit a protein kinase activity that is not stimulated by the addition of cyclic AMP or cyclic GMP. Our results suggest that a translational control system, similar to that existing in rabbit reticulocytes and other mammalian cells, is present in organisms evolutionarily far removed from mammals.

The heme-controlled translational inhibitor (HCI) of reticulocyte lysates can be activated either by a lack of by heme or, in the presence of heme, by oxidized glutathione (GSSG) and various oxidative processes. The latter activation can be prevented or reversed by NADPH or NADPH generators, such as glucose-6-phosphate (G-6-P). Since reticulocyte lysates contain a very active GSSG reductase, it was conceivable that GSSG acts by draining lysate NADPH via the reaction GSSG + NADPH + H+ in equilibrium 2 GSH + NADP+. However, removal of lysate GSSG reductase by its corresponding antibody has no effect on the activity of GSSG. This supports previous observations with lysates depleted of GSSG reductase by affinity chromatography and supports the notion that GSSG activates HCI in a more direct fashion. The role of NADPH generation in maintaining HCI in its inactive, pro-HCI form is further supported by the observation that the addition of anti-lysate G-6-P dehydrogenase antibody leads to activation of HCI in reticulocyte lysates.

Eukaryotic initiation factor 2 (elF-2) was purified from the high-salt wash fraction of Drosophila melanogaster embryos. This factor, with a molecular mass of about 90 kDa, consists of two subunits of 47 kDa and 39 kDa on dodecylsulfate/polyacrylamide gel electrophoresis. The 39-kDa subunit is phosphorylated by the hemin-controlled inhibitor of rabbit reticulocytes in a terminal fragment which can be cleaved by mild treatment with trypsin. Drosophila elF-2 is not a substrate for protein kinases capable of phosphorylating the beta subunit of elF-2 from rabbit reticulocytes. It is also shown that Drosophila elF-2 can form a ternary complex with GTP and Met-tRNAi, which can be efficiently transferred to 40S ribosomes in the presence of AUG and Mg2+. This factor is able to form a binary complex with GDP. Furthermore, purified elF-2 contains about 0.3 mol bound GDP/mol suggesting a high affinity of the factor for this nucleotide. Data supporting the notion that this affinity is increased in the presence of Mg2+, which impairs the GDP/GTP exchange on elF-2, are presented. The properties of Drosophila elF-2 suggest that this factor may be susceptible to regulation by a mechanism like that operating on rabbit reticulocyte elF-2.

Cell-free protein-synthesizing extracts prepared from the brine shrimp, Artemia salina, translate methylated mRNAs. Reovirus unmethylated mRNA is inactive as a template when methylation is prevented by the inhibitor, S-adenosylhomocysteine. A salina mRNAs from both undeveloped and developed embryos contain 5'-terminal 7-methylguanosine in an inverted 5'-5' linkage through three phosphate groups to the rest of the polynucleotide chain. Removal of the 7-methylguanosine by beta elimination converts the mRNA from an active form to one inactive in protein synthesis in extracts of A. salina or wheat germ. Extracts of undeveloped and developed embryos methylate reovirus unmethylated mRNA at the 5' ends to form 5'-terminal structures of the type, m7G(5')ppp(5')G and m7G(5')ppp(5')Gm.

Lysates from normally growing (25 degrees C) or heat shocked (37 degrees C, 45 min) Drosophila melanogaster embryos were obtained and the effect of analogues of the mRNA 5'-terminal cap, m7G(5')ppp(5')N structure and of potassium ions on their endogenous protein synthesis activity was studied. At optimal concentration of KCH3COO (75-80 mM), protein synthesis in normal lysates is strongly inhibited by cap analogues (m7GpppG, m7GDP, and m7GMP). At the same ionic conditions, heat shock lysates translate preferentially the heat shock messengers, and this translation is almost unaffected by the cap analogues. In contrast, residual synthesis of normal proteins in heat shock lysates was reduced by these compounds. By lowering the concentration of potassium ions it was possible to gradually reverse the inhibitory effect of the cap analogues in normal lysates and also to increase specifically the translation of normal mRNAs in heat shock lysates. Translation of normal mRNAs is also partial but specifically rescued by supplementing heat shock lysates with polypeptide chain initiation factors partially purified from rabbit reticulocytes. These data are consistent with the notion that the failure of normal mRNAs to be translated under heat shock conditions might be due, at least to some extent, to the inactivation of polypeptide chain initiation factor(s) involved in the recognition of the mRNA 5'-terminal cap structure.

A protein-synthesizing system consisting of ribosomes and supernatant of undeveloped Artemia saline embryos is used for assay of mRNA translation and initiation factors. This system contains the components needed for chain elongation but has low levels of mRNA and initiation factors. Exogenous mRNA is readily translated upon addition of high-speed supernatant or ribosomal salt wash of developing embryos as a source of initiation factors (IF). This requirement can be largely satisfied by a mixture of the reticulocyte factors IF-MP, IF-M3, IF-M2A, and IF-M2B. A. salina IF-M1, which is present in undeveloped embryo supernatant, can be inactivated by A. salina IF-M1 antibody without affecting translation.