This laboratory has been investigating cytoplasmic mRNA-protein complexes (mRNP) in mammalian cells. As a result of the progress made it should now be possible to investigate mRNP in a more detailed and molecular way than before. Recently, we succeeded in reconstituting functional mRNP in a rabbit reticulocyte cell-free translation system. The proteins crosslinked to mRNA by irradiating the reconstituted mRNP with UV light were indistinguishable from those crosslinked to mRNA by irradiating intact cells. Also, we showed that most, but not all, of the mRNP proteins are associated with caps or poly(A), and we have identified 2 sites in rabbit Beta-globin mRNA especially susceptible to inhibition of translation by synthetic complementary oligonucleotides. These are the 5' end near the cap and the region surrounding initiation codon. They correspond to known protein binding sites. Also, we shoed that eEF-Tu is present in mRNP. It is planned to make use of these observations and new tools in additional work on mRNP. We will attempt to identify some of the cap-associated proteins and relate them to known translation factors such as CBP I and eIF-4A. Complementary oligonucleotides will be used as probes for mRNP structure by finding out if they inhibit the binding of specific proteins to mRNA. Further experiments on eEF-Tu include finding out if it is associated with mRNA in intact cells, if it is associated with rRNA as well as mRNA, and finding out at what stage in protein synthesis it becomes associated with mRNA and rRNA. Similar experiments will be done with other translation factors. These experiments will utilize the cell-free reconstitution system and UV crosslinking methodology developed in this laboratory. A major goal will be to isolate monoclonal antibodies against a variety of mRNP proteins. They will have many uses, but a particularly important one will be to deplete lysate of the corresponding protein to find out if, and in what way, it is involved in translation. They will also be useful tools for isolating mRNP proteins, especially if no functional assay is available.
| Miseta, A; Woodley, C L; Greenberg, J R et al. (1991) Mammalian seryl-tRNA synthetase associates with mRNA in vivo and has homology to elongation factor 1 alpha. J Biol Chem 266:19158-61 |
