The ability to recognize that a genetic disease is caused by an abnormality in translational control must be based upon the definition of the mRNA structural features normally important in translation. Identification of such genetic defects may expand the understanding of pathogenesis in certain human genetic disorders such as the thalassemias and establish a scientific foundation for incorporating translational controls into the design of therapeutic approaches.
The specific aim of the proposed program is to study the genetic control of translation by defining the structural features of the two highly homologous human Alpha-globin mRNAs which result in their different translational efficiencies. The translational efficiencies of each of the two Alpha-globin mRNA species will be established in vivo and in vitro by combining accurate measurements of Alpha 1- and Alpha 2-globin mRNA levels with genetic approaches which allow quantitation of Alpha 1- and Alpha 2-globin protein synthesis. The following potential differences between the two Alpha-globin mRNA will be investigated and related to their relative translational efficiencies: a) the subcellular distrubution (sequestration in mRNP particles, association with polysomes), b) translation initiation efficiencies, and c) post-transcriptional structural modifications (5 feet capping, 3 feet polyadenylation). The translation of the two Alpha-globin mRNAs will be compared in erythroid and non-erythroid systems in vitro to detect erythroid-specific translation factors. Such factors will be characterized by reciprocal mixing experiments between the two systems. These factors will be localized to the ribosomal and/or post-ribosomal fractions, and their final isolation will be approached by RNA affinity chromatography. The 3 feet nontranslated region of the Alpha-globin mRNA will be altered by introducing deletions of primary sequences and interruptions of secondary structure to directly test the effects of this region upon translation and to specifically characterize regulatory sequences.
