|Full text PDF:||http://hdl.handle.net/10192/30621|
Piwi proteins are a subfamily of the Argonaute proteins (AGO) that interact with small RNAs to form an RNA induced silencing complexes (RISC). AGO and Piwi protein have two major protein motifs- the PAZ and PIWI. The PAZ domain binds to the 3?? end of small RNAs and the PIWI domain has an RNase H fold that can cleave a target RNA that is complimentary to their bound RNA . This study aims to develop an RNA inhibitor that binds to the PAZ domain of Xiwi - a Piwi homolog found in Xenopus. RNA aptamers were selected with a recombinant Xiwi PAZ domain using systematic evolution of ligands by exponential enrichment (SELEX). A library of RNA containing 1015 random 50 nt RNA sequences were selected for Xiwi PAZ domain binding. I selected 9 candidate RNA aptamers and generated RNAs by in vitro transcription. I characterized RNA aptamer binding ability with an electrophoresis mobility shift essay (EMSA) and in-vitro in Xenopus tropicalis egg extract to check for piRNA degradation due to displacement. EMSA showed that aptamers 2, 5, 8 and 9 bound well to the Xiwi PAZ domain. Aptamers 5 and 9 failed to displace piRNA in complex with Xiwi in X.Tropicalis egg extract. An RNA inhibitor of Xiwi serves as an important molecular tool that can be used to further characterize the functions of Xiwi. Piwi proteins are a subfamily of AGO that bind to piRNAs 20-32nt long and are important in the silencing of transposable elements. Piwi also binds to a diverse number of genes. CLIP of Xiwi (Piwi homolog found in Xenopus), and Xili was carried out by Yuliya Sytikova in order to determine genes that interacted more with Xiwi, and Xili. CLIP data was analyzed based on total CLIP counts in the open reading frame, by Dr. Chirn. The goal of this experiment is to validate the CLIP data through RIP, RT-QPCR. RIPs using X.Tropicalis stage I-IV oocyte extract was being optimized, and validated with Xiwi, and Xili western blots. RT-QPCR surveying a few genes revealed no significant difference in enrichment (% input) between positive and negative controls. Correcting and validating the CLIP data could provide huge insights regarding Xiwi, and Xili gene interactions, and silencing.