Prediction and validation of RNA structures affecting gene expression

Eukaryotic genes are typically monocistronic, containing one open reading frame (ORF)\ud that requires a 5??? modified guanine cap for expression of the gene. However, dicistronic\ud genes are a class of genes expressed with two non-overlapping ORFs per mRNA\ud transcript and require cap-independent initiation to allow expression of the downstream\ud gene. One observed mechanism involves recruitment of the ribosome to the downstream\ud gene by RNA secondary structure forming an internal ribosome entry site (IRES)\ud contained in the intercistronic region (ICR) between the dicistronic gene pair. We have\ud generated annotated alignments of dicistronic orthologs in several Drosophilid species\ud and have identified ICR regions that contain putative IRES elements using RNA\ud secondary structure prediction programs. We have sub-cloned these regions into a\ud customized dicistronic reporter gene plasmid, where a single mRNA encoding two\ud fluorescent reporter genes can be expressed in both human Hela, and Drosophila\ud Schneider (S2) cells. Comparative genomic and bioinformatics analysis of RNA\ud secondary structures along with in vitro functional assays will help us to better\ud understand the relation between primary sequence, RNA secondary structure, and IRES\ud activity. On a larger scale, study of RNA secondary structures affecting gene regulation\ud will offer insights to control of many genes, since similar mechanisms are likely used to\ud regulate more prevalent classes of genes. Understanding IRES-mediated gene expression\ud also has the potential for application to both therapeutic and infectious medicine