Harry O. Voorma

The human insulin-like growth factor II leader 1 contains an internal ribosomal entry site

Insulin-like growth factor II is a small peptide growth hormone, encoded by four mRNAs with unique 5 untranslated regions and identical coding regions. The 5 untranslated region transcribed from promoter 1 is 598 nt (leader 1). The properties of this leader 1 suggest a strong regulation of translation; the high G + C-content, the presence of an upstream open reading frame, and the length of the 5 UTR are 3 elements which prohibit efficient translation and which may modulate expression. In this paper we show that the human IGFII leader 1 harbours sequence elements that allow translation initiation to occur by internal initiation on the IGF sequence. This mode of initiation was described first for picornaviral mRNAs, that are naturally uncapped. The IGFII leader 1-dependent expression in HeLa cells was resistant to infection with poliovirus; abrogation of cap-dependent initiation by poliovirus had apparently no effect on IGFII expression. Moreover, a downstream CAT-cistron in a bicistronic construct was translated upon insertion of the leader 1 sequence. The translational properties of the IGFII leader 1 suggest that internal initiation on this leader may be modulated during proliferation or differentiation, enabling cell-stage dependent expression of IGFII.

Nerve and epidermal growth factor induce protein synthesis and eIF2B activation in PC12 cells.

The regulation of protein synthesis and of eukaryotic initiation factor eIF2B was studied in PC12 cells. An increase in protein synthesis was observed after nerve growth factor (NGF) and epidermal growth factor (EGF) treatment of PC12 cells, and this increase coincided with activation of eIF2B. Growth factor addition in the presence of the phosphatidylinositol-3′-OH kinase inhibitor wortmannin showed that both NGF- and EGF-induced protein synthesis and eIF2B activation were phosphatidylinositol-3′-OH kinase dependent. The EGF-induced stimulation of protein synthesis and activation of eIF2B was dependent upon FK506-binding protein-rapamycin-associated protein, as shown with the immunosuppressant rapamycin, whereas NGF induction was partially dependent upon FK506-binding protein-rapamycin-associated protein. The activities of two kinases that act on eIF2B, glycogen synthase kinase-3 and casein kinase II, were measured to assess their potential roles in the activation of eIF2B in PC12 cells. Inactivation of glycogen synthase kinase-3 was seen in response to both NGF and EGF and this coincided with activation of eIF2B. However, inactivation of glycogen synthase kinase-3 was not rapamycin sensitive, in contrast to the activation of eIF2B. This indicates the involvement of another protein kinase or regulatory mechanism in the eIF2B activation. Both growth factors activated casein kinase II. However, the time course of its activation and its insensitivity to wortmannin and rapamycin suggest that casein kinase II does not play a major regulatory role in eIF2B activation under these conditions.

Inactivation of eIF2B and Phosphorylation of PHAS-I in Heat-shocked Rat Hepatoma Cells

Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43u2009°C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures.

INITIATION OF EUKARYOTIC PROTEIN SYNTHESIS

The initiation of protein synthesis is defined as the sequence of events, which leads to an 80 S . MettRNA . mRNA complex. Several non-ribosomal proteins are required for the formation of this 80 S initiation complex and they are called eIF-1, -2, etc. (eukaryotic initiation factor). They are listed in table 1, with the effect they have on protein synthesis and on partial reactions thereof, as desiribed below. An initiation factor is defined as a protein which stimulates one or more of these, and only these reactions, and which is released after the completion of an 80 S initiation complex, in contrast with a ribosomal protein, which remains an integral part of the ribosome during all stages of protein synthesis. Dissociation of 80 S ribosomes is a prerequisite for the initiation of eukaryotic protein synthesis, since the initial binding of Met-tRNA occurs on a 40 S subunit and not on an 80 S ribosome. Spontaneous

Basepairing with 18S ribosomal RNA in internal initiation of translation

In concert with the translation initiation factors ‘trans‐acting’ factors function specifically during internal initiation on picornaviral mRNAs. Of these trans‐acting factors, two have been identified as the La‐protein and the polypyrimidine tract binding protein. Within the internal ribosomal entry site on the viral RNA, sequences are present that direct the ribosome to the initiation codon. We suggest that selection of the correct AUG initiation codon occurs through basepairing with a part of 18S ribosomal RNA.

THE ROLE OF eIF-4C IN PROTEIN SYNTHESIS INITIATION COMPLEX FORMATION

eIF-4C has a pronounced stimulatory effect on initiation complex formation with native 80-S ribosomes (80-Sn) as the only source of ribosomal subunits, but only a small effect when washed 40-S subunits are used. eIF-4C is accessary to eIF-3 in dissociating 80-Sn ribosomes. eIF-4C is present on 40-Sn but absent on 40-Sn dimers, which occur in preparations of native ribosomes and are as such inactive in protein synthesis. eIF-4C dissociates 40-Sn dimers into active monomers. These results can be explained by assuming that the presence of eIF-4C on 40-Sn prevents: (a) premature association with 60-S ribosomal subunits and (b) dimerisation, thus increasing the rate and extent of initiation complex formation.

THE 5' UNTRANSLATED REGION OF ENCEPHALOMYOCARDITIS VIRUS CONTAINS A SEQUENCE FOR VERY EFFICIENT BINDING OF EUKARYOTIC INITIATION FACTOR EIF-2/2B

The mechanism by which internal ribosomal binding on the picornaviral RNA takes place is still not known. An important role has been suggested for eukaryotic initiation factors eIF-4A, eIF-4B, as well as for some not yet defined trans-acting factors like p52 for poliovirus and p58 for encephalomyocarditis virus (EMCV). In this paper we describe the competition between the 5 untranslated region (UTR) of EMCV and globin mRNA for the translational apparatus in rabbit reticulocyte lysates and show that the factor that is competed for is eIF-2/2B. The EMC 5 UTR is a very strong inhibitor of globin synthesis in the rabbit reticulocyte lysate because of a 30-fold higher eIF-2/2B binding capacity. Mutations 100 to 140 nucleotides upstream of the initiation codon led to a decreased efficiency to initiate translation and to a decreased ability to inhibit globin mRNA translation. The results suggest an important role for eIF-2/2B binding in EMC RNA translation and therefore in internal initiation.

Role of the 3' untranslated region of baculovirus p10 mRNA in high-level expression of foreign genes

The p10 gene of Autographa californica nucleopolyhedrovirus has two putative AATAAA polyadenylation signals. The downstream signal is used predominantly, as was determined by analysing 3 cDNA ends. This downstream motif is followed by a GT-rich sequence, known to be important for efficient polyadenylation in mammalian systems. To analyse the importance of polyadenylation for p10 gene expression, recombinant viruses with altered 3 untranslated regions (UTRs) were tested using chloramphenicol acetyltransferase (CAT) as a reporter. Surprisingly, after inactivation of the downstream AATAAA motif, CAT expression remained at the same high level as observed with a wild-type 3 UTR. Polyadenylation occurred 24-28 nucleotides further downstream, probably due to an ATTAAA sequence motif. Replacing the p 10 3 UTR with the SV40 early terminator sequence as part of an hsp70-lacZ-SV40 gene cassette, which is commonly used in baculovirus expression vectors, resulted in a reduction in reporter gene expression. Polyadenylation occurred far more efficiently in the original p10 3 UTR than in the SV40 terminator sequence, as was shown by testing the SV40 terminator separately. These results indicate that in order to obtain high levels of foreign gene expression, vectors that provide a wild-type p10 3 UTR are to be preferred over those containing the hsp70-lacZ-SV40 gene cassette. Comparison of the p10 genes of various baculoviruses showed the presence of at least one AATAAA or ATTAAA motif in combination with a GT-rich sequence in the 3 UTR, suggesting an evolutionary conservation of these two elements, thereby maintaining the high level of p10 gene expression.

Initiation of protein synthesis in neuroblastoma cells infected by Semliki forest virus A decreased requirement of late viral mRNA for eIF-4B and cap binding protein

Semliki Forest Virus causes shut-off of protein synthesis in its mammalian hosts [ 1,2]. Usually, this shutoff takes place 3-5 h post-infection [2]. In neuroblastoma cells from mouse such an event leads to the exclusive production of late viral (= structural) proteins (e.g., ~97, ~62, El and capsid protein, see [2--41). These proteins are synthehized as a large precursor from a sub-genomic template of 26 S [ 1 ,.S] an mRNA which appears late in infection and is identical to the 3’-terminal part (-1/3rd) of the viral genomic RNA of 42 S [6,7]. This 42 S mRNA serves as a template early in infection for the synthesis of non-structural proteins [2,4]. The genes for these proteins are located at the 5’-terminus of this messenger, whereas the genes for the structural proteins, which are found at the 3’-part of 42 S are silent (see above, [2]). At a late stage of infection, 26 S mRNA is the only messenger recognized by the protein synthesizing machinery, despite of the fact that still considerable amounts of 42 S and host mRNAs are present in the infected cell [1,%31. High salt washes of ribosomes (containing the initiation factors of protein synthesis), isolated from neuroblastoma cells at a late stage of infection by SFV, have mostly lost the ability to support the trsnslation in ‘in vitro’ systems of hostand early viral mRNA, although they are still highly active with viral lateand EMC mRNA [9]. Addition to systems programmed with hostand viral early mRNA, of purified initiation factors eIF4B (M, 80 000, [9,10]) and cap binding protein (CBP, MI 24 000, [9,1 l]), but none of the others significantly increases the activity of these infectionexposed crude initiation factors. These fmdings indicate that the lesion in the protein synthesizing machinery has occurred at the level of initiation factors eIF4B and CBP, although the precise mechanism by which these factors are incapacitated remains, at present, unclear. Here we substantiate the ability of purified eIF4B and CBP to stimulate the activity of infectionexposed crude factors in protein synthesis in an ‘in vitro’ system programmed with early viral mRNA. We explore why the SFV-induced blockade of eIF4B and CBP activity results in a preferential recognition of late SFV mRNA by the protein synthesizing machinery in infected cells. We show that optimal translation of 26 S mRNA requires a 2-4-times smaller amount of eIF4B and CBP when compared to hostand 42 S mRNA. Such a low requirement for eIF4B and CBP results in a relatively undisturbed translation of viral late mRNA in infected cells in spite of the fact that these factors are gradually inactivated. The reason for the decreased need for eIF4B and CBP, factors which are thought to be involved in the recognition of the 5’-terminal cap structure of mRNAs [ 12-141 is not based on a decreased importance of the cap-structure of 26 S mRNA, since chemically decapped 26 S RNA almost completely looses its capacity to serve as a template in pH 5 systems.

Phosphorylation of the eIF4E‐binding protein PHAS‐I after exposure of PC12 cells to EGF and NGF

PHAS‐I or the eIF4E‐binding protein 1 regulates the cap‐binding activity of eIF4E by sequestering eIF4E. Binding of eIF4E to PHAS‐I is regulated by phosphorylation of PHAS‐I. PC12 cells were used to study the signal transduction pathway leading to phosphorylation of PHAS‐I. Both EGF and NGF induced phosphorylation of PHAS‐I. Wortmannin, a PI‐3 kinase inhibitor, staurosporine, a PKC inhibitor, and rapamycin, a FRAP inhibitor all blocked the phosphorylation of PHAS‐I. Of the three inhibitors, only wortmannin was able to inhibit MAPK phosphorylation. This excludes a role for MAPK in NGF‐ and EGF‐induced PHAS‐I phosphorylation in PC12 cells. Apparently, PHAS‐I was phosphorylated in a PI‐3 kinase‐, PKC‐, and FRAP‐dependent manner after EGF or NGF stimulation. Only PI‐3 kinase and FRAP are involved in the regulation of the basal level of PHAS‐I phosphorylation.