9601148 Woodson Post-transcriptional processing of RNA is a fundamental step in eukaryotic gene expression and consequently of basic importance to biological processes such as embryonic development and disease. A central problem in RNA processing is understanding how proteins contribute to the formation of catalytic RNA structures. We will use protein-dependent splicing of a group I intron as a model for the folding and assembly of protein-RNA complexes in vivo. A protein that accelerates splicing of a group I intron from Physarum nuclear pre-ribosomal RNA has been biochemically identified in nuclear extracts of Physarum polycephalum. This protein is the first example of a nuclear group I splicing factor. An advantage of this simple system for biochemical studies is that the intron RNA is able to self-splice in the absence of protein, enabling a direct comparison of protein-dependent and protein-independent splicing in vitro. In addition, we have previously established a bacterial system for expression and splicing of group I introns in pre-ribosomal RNA. Consequently this system is well suited for future investigations of protein-RNA interactions in vivo and will be directly relevant to processing of pre-ribosomal RNA. Preliminary work has shown that this Physarum splicing factor primarily recognizes a tRNA-like structure that is not part of the catalytic core of the intron. As maturase proteins that facilitate group I splicing are so far known to predominantly bind their cognate intron core, our observation suggests a novel mode of group I protein-dependent splicing. The minimal binding site and essential recognition elements within this tRNA-like structure will be determined using chemical modification and ribonuclease protection, and site-directed mutagenesis. Models of protein-facilitated splicing will be evaluated by determining the dependence of in vitro splicing kinetics on variables such as protein concentration, temperature and time of preincubation. Protein-induced changes in the structure of the catalytic core will be determined by comparing the pattern of Fe-EDTA-dependent hydroxyl radical cleavage of intron RNA in the presence and absence of protein. Intermediates in the complex assembly pathway will be separated by native gel electrophoresis. The Physarum splicing factor will be cloned and expressed as a fusion protein in E. coli. cDNA libraries will be screened by hybridization to probes derived from peptide sequences or by complementation of splicing in bacteria. Biochemical characterization of the cloned protein will permit more detailed analysis of protein function and investigation of whether this protein binds other cellular RNAs. Development of bacterial (or yeast) expression systems will lead to further work on in vivo splicing of group I introns and enable us to dissect the roles of non-specific and specific RNA-binding proteins in the processing of pre-ribosomal RNA. %%% Most genes in higher organisms contain non-coding sequences, or introns, that are removed after the gene is transcribed into RNA. This process, which is called "splicing", is an essential step in the normal expression of genes in human and other organisms. Defective splicing can lead to disease or to developmental disorders. Correct splicing is determined by the sequence and structure of the RNA itself, and also by interactions between RNA and proteins. We have isolated a protein that promotes correct splicing of a group I intron in pre-ribosomal RNA of the slime mold Physarum polycephalum. This system will be used as a model for investigating how proteins enhance splicing reactions. The points of interaction between the RNA and the protein will be determined, and the way in which the protein alters the three-dimensional structure of the RNA investigated. In the future we will determine whether this protein also regulates expression of other genes. These experiments will lead to a better understanding of how more complex splicing systems in human genes are controlled. ***
