UC Santa Cruz

The spliceosome is an important molecular complex that converts pre-mRNA to mRNA. It removes non-coding regions of RNA known as introns and connects the exons through a two-step chemical reaction. The spliceosome is made up of five snRNPs and dozens of proteins which enter and exit the spliceosome at different points of the splicing cycle. Many of the interactions between spliceosome components remain a mystery. The goal of my research is to create a tool for the lab which will allow us to further investigate the interactions of proteins within the splicing cycle. To do this I adapted a protocol developed by Khandelia et. al. to create adherent HeLa cell lines that express tagged spliceosome proteins. These cells will allow us to perform immunoprecipitations, so we can analyze the sub-complexes that are pulled down with the tagged protein. I successfully tagged was RBM17. It was confirmed that immunoprecipitation of tagged RBM17 pulled down sub-complexes. This will allow the lab to further investigate the interactions that RBM17 makes with other spliceosome proteins. I also tested the SF3B4, but determined that HeLa cells will not express a tagged version of this protein. The other part of my research investigated the effects of splicing inhibitory drugs in-vivo. I focused on the natural product herboxidiene, which targets the SF3B complex of the spliceosome. I tested how various chemical modifications changed the effectiveness of this drug on inhibition of cell growth. This research builds on a paper recently published by Gamboa Lopez et. al. that focused on the drug’s effects on cell-free splicing. I found that different chemical modifications can modulate the strength of the drug and that some of the results contradicted the cell-free data. These studies are important as this drug is a potent inhibitor of cancer cell growth and a further understanding could one day lead to this drug’s application as a cancer therapeutic.