Bacterial small noncoding RNAs affecting gene expression in eukaryotes

Wolbachia is a highly studied bacteria found in over 70% of insects around the world. Based on recent research in Dr. Hagen’s lab he suggests that Wolbachia bacteria causes disruption in host’s reproduction abilities due to small noncoding RNAs binding to the host's DNA. This phenomenon is known as cytoplasmic incompatibility (CI) and theoretically affects the way the host expresses genes. Similar research conducted by a fellow undergraduate student suggests the small RNAs produced by Wolbachia bind to Drosophila protein coding genes. This will cause a derangement in the protein expression of the Drosophila helping to explain cytoplasmic incompatibility. This project has allowed for further investigation into the relationships between bacterial small noncoding RNAs with protein coding genes in insects. Previous research in the Hagen Lab has shown evidence that several small noncoding RNAs could have binding potential to protein coding genes. The RNA library pools from Drosophila cells and Drosophila cells infected with Wolbachia have been sequenced and analyzed. The analysis shows several potential candidate 3’UTR sequences. The plasmid used for this project is the pAc5-DsRed2-Sv40 plasmid, which was constructed by a fellow member of the lab from using both the pHd-DsRed plasmid and the act5c plasmid. The pAc5 plasmid contains an ACT5C promoter, the DsRed fluorescent gene, and an ampicillin resistance gene. Different 3’UTR sequences have been added to the plasmid to compare hybridization rates of the small noncoding RNAs. Each plasmid type will be transfected into Drosophila cells, one set infected and the other uninfected. If the small noncoding RNAs of the Wolbachia hybridize to the Drosophila protein coding genes then it is predicted to see an absence or decrease in the fluorescent protein expression. We will be able to recognize the small RNA as nontargeting if the small RNAs are not hybridizing or are low in numbers. With the use of different 3’UTR sequences we will be able to strengthen hybridization of different small RNAs to the Drosophila protein coding genes, reducing the amount of non targeting RNAs. This project aims to uncover the genetic interactions between bacteria and their eukaryotic hosts, helping us understand how these relationships impact human and animal health