This laboratory is investigating proteins associated with cytoplasmic mRNA and its nuclear precursor, hnRNA, in animal cells with the aid of ultraviolet-light induced crosslinking. It was previously found that mRNA-associated proteins differ in composition from hnRNA associated proteins. Furthermore, they associate in the cytoplasm with previously existing mRNA and exchange there with a pool of proteins not associated with mRNA. These observations suggest that the cytoplasmic mRNA-associated proteins function in protein synthesis. Recently, it was found that at least one protein, p29, seems to be associated with mRNA only during translation. Furthermore, when initiation of translation is inhibited with actinomycin D and heat shock, the amount of mRNA-associated p29 decreases, whereas the amount of another protein of slightly lower mobility, p34, increases. This laboratory is also investigating proteins specifically associated with mRNA caps with the aid of UV crosslinking and an antibody to caps. It is now planned: i) To develop novel immunological probes for RNA-protein interactions by making antibodies to nucleosides. These will be used to probe Western blots of UV-crosslinked RNAase-digested mRNA-associated proteins. Only proteins crosslinked to RNA will react. The antibodies will be used as a substitute for radioactive labeling procedures. ii) To reconstitute mRNA-protein interactions in a mRNA-dependent reticulocyte cell-free translation system. This will be done to test the hypothesis that the proteins becoming associated with mRNA in the course of translation are the same ones associated with mRNA in intact cells. iii) To identify proteins associated in intact cells with the caps of hnRNA with the aid of UV crosslinking and antibody to caps. This will be a necessary step towards the complete description of hnRNA-associated proteins. iv) To further investigate mRNA-associated proteins p29 and p34, their relationship to one another and to initiation factor eIF-2, their possible phosphorylation, and their involvement in initiation of protein synthesis. v) To investigate proteins associated with sea urchin histone mRNA. The histone mRNA-associated proteins will crosslinked to the RNA by irradiating eggs and embryos, then isolated by hybridizing to cloned DNA. The proteins will be analyzed by standard procedures. The goal is to find out to what extent different kinds of mRNA are associated with different proteins, and, eventually, to investigate the mechanism of mRNA masking (translational control) in development.
| 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 |
