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Show Genetic Engineering Tools for Understanding Complex Biological Systems Tracy Zundel, Jennifer Bait and Professor Katharine Ullman Department of Oncological Sciences siRNA Blocks Protein Expression B Rescuing RNA From siRNA DNA Cells are divided into two main compartments, the nucleus and the cytoplasm. Nuclear pores are large, complex protein structures that play a major role in regulating transport of molecules into and out of the nucleus, where the genetic code of the cell resides. Learning how these pores work will allow us to understand more about normal cell function, as well as situations in which the pore functions abnormally, such as cancerous cells. (1) Nuclear pore complexes are composed of approximately thirty components, each of which is present multiple times. In order to understand how these pores operate, the individual components must be studied. The pore component Nupl53 is located on the nuclear side of the pore and is known to be important to proper pore function. Exactly how Nupl53 works, however, is not known. (2) Cells use RNA as an intermediate to communicate genetic information encoded in the DNA template. Once RNA has been made in the nucleus, it is exported to the cytoplasm where it, in turn, serves as a template for protein synthesis. (3) One challenge to better understanding intricate biological machines such as nuclear pores is the creation of a system that can be controlled and modified. Researchers have developed a new technique called RNA interference (or RNAi) that is an important tool in dissecting cellular function. RNAi prevents the production of specific proteins by using small, interfering pieces of RNA called siRNA (silencing RNA). siRNAs bind to a specific site on a particular RNA. When siRNA binds, it directs degradation of the RNA, and thus no protein is synthesized. We are taking advantage of this technique to target the RNA that codes for Nupl53 for degradation. (4) However, others have shown that if a cell has no Nupl53, it dies. So, although this tells us Nupl53 is important, it does not answer the question of how it works. The key to answering this question is to assess pore function when Nupl53 has been altered rather than when Nupl53 is completely absent. Genetic engineering techniques allow us to replace the protein targeted by the siRNA by introducing DNA into the cell that encodes a slightly modified version of the target RNA. By slightly altering the sequence or code of the RNA, one can design an RNA that overall still codes for the same protein but is unable to be targeted by the siRNA. In this way, the original RNA encoding normal protein is degraded, but a replacement RNA is present. This rescues the cell from death and gives us a system in which to test the effect of making changes in the protein of interest. (5) By introducing other changes into the si-RNA-resistant template, altered versions of the protein can be generated. Now, instead of replacing the original protein with normal protein, it is replaced with an altered protein. By inactivating specific sub-regions of a protein, we can test how the protein works. (6) My project is to design and build DNA constructs that can be used to study Nupl53 in this manner. In the case of Nupl53, we hope to learn what parts of this pore component help different types of cargo move through the pore. Ultimately, we hope to learnhow to control aspects of nuclear pore function in ways that will be useful in fighting cancer. |