Very little is known regarding eukaryotic messenger RNA higher order structure considering its importance during gene expression. Determining the relationship of mRNA structure to translational initiation efficiency, recruitment and repression of mRNA populations, and turnover is paramount to understanding these processes at the molecular level. We are now determining the secondary structures of mouse alpha and beta globin mRNAs. The mammalian alpha and beta globin mRNAs represent a well documented system, whereby beta globin mRNA initiates translation at a rate 50% faster than alpha mRNA within a reticulocyte. Both mouse globin mRNAs are extremely stable, with half-lives of greater than 60 hours. These mRNAs can be purified in their native form from reticulocytes in large enough quantities necessary for structure analysis and determination of RNA-protein interactions. Messenger ribonucleoprotein particles (mRNPs) in both the free state and within polyribosomes will be probed chemically with diethylpyrocarbonate, dimethylsulfate, and hydroxymethylpsoralen to determine both the single- and double-stranded regions of the molecules. Secondary structure models will be derived by combining comprehensive biochemical and phylogenetic data with computer analysis. The identity of the proteins we have already determined to be associated with mouse globin mRNA by photo-induced cross-linking will be ascertained by immunoblotting. The points of contact between the cross- linked proteins and the mRNAs will be mapped by specific immunoprecipitation of T1 ribonuclease digested mRNP followed by sequence analysis of the cross-linked RNA oligomer. Establishing the identity and site of attachment of these proteins with respect to the derived structure models, should generate a view of the architecture of the globin mRNAs in both the free and translationally active states. Knowledge of the definitive sequence and structural features recognized by these proteins should help reveal their specific roles within the cell. Once secondary structure models for both mRNAs are established, site- directed mutagenesis could be carried out in the future so to define the specific sequence and structural features important to their function.
| Robertson, L S; Fink, G R (1998) The three yeast A kinases have specific signaling functions in pseudohyphal growth. Proc Natl Acad Sci U S A 95:13783-7 |
| Rairkar, A; Lockard, R E (1988) Characterization of mouse reticulocyte free globin mRNP. FEBS Lett 241:73-8 |
| Lockard, R E (1987) Identification of the proteins in direct contact with duck globin mRNA. FEBS Lett 219:410-4 |
| Lockard, R E; Currey, K; Browner, M et al. (1986) Secondary structure model for mouse beta Maj globin mRNA derived from enzymatic digestion data, comparative sequence and computer analysis. Nucleic Acids Res 14:5827-41 |
