This work will investigate the function of an endogenous antisense RNA in regulating per clock gene expression and, therefore, behavioral rhythms in the giant silkmoth. The full-length cDNA for the antisense transcript will be determined by first mapping a minimal sequence by ribonuclease protection assay followed by 5' and 3' RACE. This will delineate the possible regions of hybridization that might occur between these two transcripts in vivo. The functional importance of RNA duplex formation will then be investigated by administering exogenous antisense oligonucleotides designed to disrupt RNA-RNA interactions while examining alterations in per mRNA and protein oscillation as well as behavioral rhythms. Similar experiments utilizing antisense oligonucleotides will be done to attenuate the translation of any proteins that may be encoded by the novel antisense cDNA. Duplex formation between the sense and antisense per transcripts and the disruption of these by exogenous oligonucleotides will be confirmed by modified ribonuclease protection assays. These studies will lend insight into the molecular mechanisms of circadian behavior and contribute to our understanding of sleep disorders and circadian-based susceptibility to disease.
| Gotter, Anthony L; Suppa, Christine; Emanuel, Beverly S (2007) Mammalian TIMELESS and Tipin are evolutionarily conserved replication fork-associated factors. J Mol Biol 366:36-52 |
