Ribosomal scanning on the highly structured insulin-like growth factor II-leader 1.

The complex architecture of human insulin-like growth factor (IGF) II-leader 1 of 592 nucleotides (nt), with one open reading frame (ORF), and the potential to fold into stable structures makes efficient linear ribosomal scanning difficult to comprehend. Indeed, leader 1-driven reporter expression is low in rabbit reticulocyte lysate. Contrarily, leader 1 is very efficient in cells.

Vector design for optimal protein expression.

Many DNA constructs are generated for protein expression studies. Translational properties and mRNA stability are crucial aspects that have to be accounted for during DNA construction. An optimized vector for protein overexpression studies is described considering elements in the mature mRNA that influence translatability and stability.

Sequence and translation initiation properties of the xenopus TGFbeta5, PDGF-A, and PDGF-alpha receptor 5' untranslated regions.

The properties of the architecturally complex Xenopus laevis TGFbeta5, PDGF-A and PDGF-alpha receptor 5'UTRs were investigated. 5' extended cDNAs were obtained by 5'RACE, resulting in long 5'UTRs (478-710 nt) with multiple upstream AUGs (3-13), andthe potential to fold into stable structures. Injection studies suggested that the cloned PDGF-alphaR 5'UTR contains an intron.

Controlled translation initiation on insulin-like growth factor 2-leader 1 during Xenopus laevis embryogenesis.

A number of growth factors, whose spatio-temporal expression is essential for embryonic development, are encoded by mRNAs with a complex 5'UTR. Human insulin-like growth factor 2 mRNA contains a long (592 nucleotides) 5'UTR (IGFl1) with one upstream open reading frame and stable stem-loop structures, elements which might be important for controlled translation. To investigate whether these unusual features of IGFl1 can control translation initiation during embryogenesis, we examined the initiation efficiency on this 5'UTR during development of Xenopus laevis.

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.

Regulation of translation initiation factors by signal transduction.

Regulation of eukaryotic translation initiation is a process that requires collaboration between multiple proteins. The cap-binding factor eukaryotic initiation factor (eIF)4E, its binding protein 4E-BP1, and the guanine-nucleotide-exchange factor eIF2B play important roles in the regulation of the rate of protein synthesis. This review describes the regulation of the activity of these three proteins and the signal-transduction pathways involved therein.

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.

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 43 degrees C or higher while protein synthesis is already affected at milder heat shock temperatures.

Translational properties of the untranslated regions of the p10 messenger RNA of Autographa californica multicapsid nucleopolyhedrovirus.

Protein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mRNAs. Therefore, a novel aspect of the baculovirus expression system was studied: initiation of translation of very late mRNAs of Autographa californica multicapsid nucleopolyhedrovirus. The untranslated regions (UTRs) of the p10 mRNA of this baculovirus were studied by in vitro translation and after transfection into Spodoptera frugiperda insect cells.

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 elF4E 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.

Phosphorylation state of the cap-binding protein eIF4E during viral infection.

The eukaryotic translation initiation factor eIF4E, the cap-binding protein, seems to play an essential role in the establishment of the host shut-off after viral infection. Infection with adenovirus and influenza virus caused dephosphorylation of eIF4E and an involvement of a viral protein was suggested. In this report, we studied several other viruses for their ability to change the phosphorylation state of eIF4E, and we looked for the mechanism of eIF4E dephosphorylation.

Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA.

Foot-and-mouth disease virus (FMDV) RNA utilizes two in-frame initiation codons to produce two precursor proteins with identical carboxy termini. The 5' untranslated region (5'UTR) directs the ribosome to internal sequences without the need for a cap structure as used in host mRNAs. The FMDV 5'UTR was cloned upstream of the reporter gene chloramphenicol acetyltransferase (CAT) in order to study the selection of initiation site and to facilitate quantification of the translation products.

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.

Phosphorylation of eIF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells.

Mitogenic stimulation of protein synthesis is accompanied by an increase in eIF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition.

The amino acid sequence of eukaryotic translation initiation factor 1 and its similarity to yeast initiation factor SUI1.

Eukaryotic initiation factor eIF-1 was purified from rabbit reticulocytes. Amino acid sequence analysis revealed that the protein contained a blocked amino-terminus. After cleavage with the endoproteinase Asp-N, three peptides were sequenced.

Translation initiation on the insulin-like growth factor II leader 1 is developmentally regulated.

The majority of cellular mRNAs have relatively short and unstructured 5' untranslated regions (UTRs) that allow efficient translation, such as the beta-globin mRNA. An exception to this rule is the group of growth factor mRNAs which, in general, have long 5' UTRs with a high G + C content. An example is insulin-like growth factor II (IGF-II), which is encoded by four mRNAs, arising from four different promoters.

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.

Initiation of protein synthesis in eukaryotes.

The study of the regulation of initiation of protein synthesis has recently gained momentum because of the established relationship between translation initiation, cell growth and tumorigenesis. Therefore much effort is devoted to the role of protein kinases which are activated in signal transduction cascades and which are responsible for the phosphorylation of a number of initiation factors. These specific factors are mainly involved in the binding of messenger RNA to the 40S ribosome, a process that makes the unwinding of the 5' untranslated region necessary.

Binding of eukaryotic initiation factor-2 and trans-acting factors to the 5' untranslated region of encephalomyocarditis virus RNA.

The encephalomyocarditis virus 5' untranslated region (EMC 5' UTR) has a binding site for eukaryotic initiation factor eIF-2. Mutations in the 3' end or deletion of the 5' end of the internal ribosomal entry site had a negative effect on the binding of eIF-2 to the EMC 5' UTR. The binding of eIF-2 to the mutant 5' UTRs was completely inhibited by the addition of competitor tRNA.

Epidermal growth factor stimulates phosphorylation of eukaryotic initiation factor 4B, independently of protein kinase C.

We demonstrate that exposure of human epidermoid carcinoma A431 cells to epidermal growth factor (EGF) results in phosphorylation of eIF-4B within minutes after addition of EGF. The EGF-induced phosphorylation of eIF-4B is not caused by the EGF receptor tyrosine kinase itself, since no tyrosine-phosphorylated eIF-4B could be detected upon immunoprecipitation using an anti-phosphotyrosine antibody. Enhanced phosphorylation of eIF-4B was also detected upon exposure of the cells to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), suggesting that eIF-4B may be a substrate of PKC.

Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5'-untranslated regions of Semliki Forest virus RNA is related to translational efficiency.

Chimaeric chloramphenicol acetyltransferase (CAT) mRNA, containing the leader sequences of genomic 42S RNA and subgenomic 26S RNA of Semliki Forest virus (SFV) were synthesized by in-vitro transcription. These transcripts were translated with different efficiencies, as the authentic mRNA in SFV-infected cells. Therefore, they can be used as model mRNA species to study the mechanism underlying SFV-directed shut off of host protein synthesis.

Dependence of the adenovirus tripartite leader on the p220 subunit of eukaryotic initiation factor 4F during in vitro translation. Effect of p220 cleavage by foot-and-mouth-disease-virus L-protease on in vitro translation.

The adenovirus tripartite leader (TPT) 5' untranslated region (5'UTR) allows translation in poliovirus-infected cells, in which the p220 subunit of eukaryotic initiation factor 4F is degraded. This p220-independent translation was investigated by measuring in vitro translation in a reticulocyte lysate of a reporter gene, chloramphenicol acetyltransferase, coupled to the TPT 5'UTR. The p220 subunit was degraded by translation of a foot-and-mouth-disease L-protease construct.

RNA unwinding by eukaryotic initiation factor 4A and nucleotide modification.

Unwinding of double-stranded RNA by nuclear helicases can lead to modification of adenosine-residues, resulting in inosine. During initiation of protein synthesis the 5' untranslated region of an mRNA is unwound by eukaryotic initiation factors (eIF) -4A and -4B. In this work we investigated the possible nucleotide modification after unwinding by eIF-4A and eIF-4B of in vitro synthesized, labeled RNA.

Interaction of protein synthesis initiation factors with the mRNA cap structure.

The mechanism of mRNA recognition by proteins interacting with the mRNA cap structure was investigated by photochemical cross-linking of proteins with 32P-labelled reoviral RNAs. Using ribosomal washes as a source of eukaryotic protein synthesis initiation factors, we identified the well-known cap binding proteins eIF-4B and -4E, but eIF-2 and eIF-3 as well. The interplay of purified eIF-4A, -4B, and -4F was studied in relation to ATP dependence and cap analogue sensitivity of cap binding.

Eukaryotic initiation factors-4E and -4F stimulate 5' cap-dependent as well as internal initiation of protein synthesis.

Two mechanisms of initiation of protein synthesis are known. The 5' cap-dependent model requires the activity of cap-binding eukaryotic initiation factors (eIF)1-4E and -4F, inducing unwinding of mRNA secondary structures. The internal initiation model is 5' cap-independent and requires a ribosomal entry site formed by higher order structures of the mRNA.