The objective of the planned research is to elucidate the structures of three RNA elements critical in pre-mRNA splicing reactions. Little is known about the role RNA structure plays in the many biological processes in which it participates. Pre-mRNA splicing is an excellent system in which to study the relationship between RNA structure and function, because RNA is involved not only as the substrate, but also in structural and in catalytic roles. The most familiar form of pre-mRNA splicing is cis-splicing, the intramolecular joining of two exons with the excision of an intron. Mutations in splice donor or acceptor regions can result in the use of """"""""cryptic"""""""" splice sites; a number of genetic diseases stem from altered gene products derived from aberrant splicing. Trans-splicing, which involves the transfer of a short 5' spliced leader (SL) sequence to a separate pre-mRNA transcript, utilizes the same chemistry and shares certain other structural characteristics with the self-splicing introns and with cis-splicing. Since many of the organisms employing trans-splicing are responsible for parasitic disease, knowledge of SL RNA structures presents a unique target for drug design.
Specific aims are to determine high resolution structures of motifs involved in cis- and trans-splicing reactions. Particular interest is focused on the following internal loop/bulge motifs. 1) The structure of the entire first stem loop of the SL1 RNA of C. elegans will be determined. The stem contains an asymmetric internal loop known to bind to a specific protein. This project represents an extension of previous structural studies. 2) The bulged region formed by the pairing between the yeast spliceosomal intron branch site and the U2 snRNA will be investigated. The adenine residue bulged out by this pairing is the nucleophile in the first step of splicing. 3) A third structural target is the bulged region formed by the pairing of the spliceosomal U2 and U6 snRNAs, which is essential to both steps in the pre-mRNA splicing reaction. RNA fragments will be studied by a combination of multidimensional homonuclear and heteronuclear NMR techniques and biochemical methods. Information obtained from these studies will provide new information about the structures formed by biologically active RNA molecules and the basis for their activity.
