Biomedical subjects

M A Kasperaitis

Publications and source records attributed to M A Kasperaitis.

5 recordsLinked to original sources

PubMed1995

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. The obtained partial sequences were identical to sequences of SUI1ISO1, the human homologue of the yeast translation initiation factor SUI1. The SUI1 gene product was identified as a protein involved in the recognition of the protein synthesis initiation codon. A similar mode of action has been suggested for eIF-1.

Amino Acid Sequence↗

PubMed1994

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. Transcripts having the human IGF-II leader 1 are only expressed in adult liver where IGF-II protein synthesis is solely under direction of this 5' UTR. We investigated the translational efficiency in vitro of this 5' UTR, linked to the chloramphenicol acetyltransferase (CAT) encoding region. As expected from the primary structure of IGF-II leader 1, translational efficiency was very low compared with beta-globin 5' UTR-CAT mRNA. Addition of cell extract from undifferentiated P19 embryonal carcinoma (EC) cells preferentially stimulated translation of an IGF-II 5' UTR RNA construct. No translational stimulation was found when cell extract from differentiated P19 EC cells was added. In contrast with the beta-globin 5' UTR, translation initiation on the IGF-II 5' UTR was not dependent on the presence of a cap structure. The results imply that only in undifferentiated P19 EC cells and not in their differentiated derivatives is a factor present that specifically stimulates IGF-II RNA translation, thereby suggesting translational regulation of IGF-II production during early embryonic development. A mechanism for translation initiation on the 5' UTR of IGF-II is discussed.

Adenoviridae↗

PubMed1993

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. However, down-regulation of PKC did not influence the EGF-induced eIF-4B phosphorylation, which indicates that eIF-4B is phosphorylated by an as yet unknown kinase, activated early in the EGF-induced signal transduction cascade.

Enzyme Activation↗

PubMed1992

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. The interaction of translation initiation factors with the 5' cap structure was studied. Transcripts prepared in vitro using T7 RNA polymerase were capped and methylated posttranscriptionally with [32P]-GTP and S-adenosyl-L-methionine to yield cap-labelled mRNA species. Irradiation with ultraviolet light of 26S CAT and 42S CAT transcripts, together with crude rabbit reticulocyte initiation factors, resulted in the cap-specific cross-linking of eukaryotic initiation factors (eIF) eIF-4E and eIF-4B. The relative binding efficiency of these two factors to the cap structure of the various transcripts was, however, markedly different; the cap structure present in 26S CAT mRNA interacted efficiently with cap-binding proteins, whereas the cap structure of 42S CAT mRNA hardly bound to these proteins. Comparable results were obtained under competitive conditions. Data are presented that the secondary structure close to the 5' cap structure determines the efficiency of recognition of the mRNA by these initiation factors. Using a chemical cross-linking assay, it was demonstrated that eIF-4F, and also eIF-4E, differentially interacted with the cap structure of the various transcripts. The data are discussed with respect to the possible mechanisms involved in SFV-induced shut off of host cell protein synthesis.

Base Sequence↗

PubMed1991

Evidence that eukaryotic initiation factor (eIF) 2 is a cap-binding protein that stimulates cap recognition by eIF-4B and eIF-4F.

We studied the mRNA-binding properties of eukaryotic initiation factor (eIF) 2. This Met-tRNA-binding factor interacts with the cap structure of reoviral mRNA in an ATP-independent manner. Both the beta- and gamma-subunit of eIF-2 are involved in the UV-induced cross-linking of eIF-2 to the cap. The interaction of eIF-2 with a messenger is sensitive to the cap analogue 7-methyl-guanosine 5'-triphosphate as measured by cross-linking and by mRNA retention on nitrocellulose filters. The cap-binding property of eIF-2 does not conflict with the current mRNA-binding model of initiation factors eIF-4A, -4B, and -4F: cross-linking of eIF-4E and of eIF-4B is stimulated by eIF-2. The eIF-2-mediated increase of eIF-4E interaction results in a decrease of the cross-linking of the beta- and gamma-subunits of eIF-2. The presence of GTP in the cross-linking assay interferes with the interaction of eIF-2 with the cap structure but does not inhibit the eIF-2 stimulated eIF-4E and -4B cross-linking. These observations indicate a role for eIF-2 in the mRNA recognition.

Animals↗