The long-term objective of this research is to understand the molecular mechanisms governing messenger RNA trafficking in eukaryotes. This process which encompasses mRNA sorting, transport and localization is emerging as a means of imparting specificity to cells and subcellular regions. It functions in the establishment of axial polarity in development and has been implicated in the determination of polarity within differentiated cells. All indications are that RNA localization is likely to take its place alongside splicing, polyadenylation, stabilization and translation as a major regulatory step in the expression of some mRNAs and that the act of localizing an RNA may have profound implications for the other processes that affect a transcript during its lifetime. A multi- disciplinary approach will be taken to the dissection of the trafficking process with a heavy emphasis on techniques from molecular biology and biochemistry. Extensive use will be made of synthetic RNA transcripts, cellular extracts and the battery of techniques used to successfully investigate RNA-protein interactions in other steps of RNA biogenesis. As a model substrate for these experiments, the investigator has selected the bicoid mRNA, which is localized post-transcriptionally within the oocyte and ultimately to the anterior tip of the Drosophila embryo. This system has the advantages of being genetically-defined and biochemically accessible. Immediate objectives are to: 1) identify bicoid's discrete, cis-acting RNA localization signals by sequence comparison of localized RNAs and by their physical interaction with the three genetically-defined factors for bicoid mRNA localization and 2) employ these defined localization signals to characterize and isolate the additional trans-acting localization factors which recognize them. While RNA localization is clearly complex, this targeted approach will focus the investigation on very specific aspects of the process. Analyses will include examination of an evolutionarily conserved 9 nucleotide motif common to several localized messages as well as definition of the cis-acting localization signals in the bicoid 3' UTR which interact with the three, previously mentioned, genetically- defined, trans-acting localization factors. The investigation outlined here may contribute substantially to the detailed understanding of the components of the localization machinery and their molecular interactions. This will facilitate definition of the fundamental principles underlying the important process of mRNA trafficking as well as delineate the specificity used to localize molecules to different regions of a single cell.
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