Cellular Fractionation to Characterize the Interaction of Nucleolin with Alpha-COP

Background and Hypothesis:The goal was to further characterize the interaction between Nucleolin alpha subunit of COPI coatomer complex. contains a C-terminal dilysine motif, which mediates interactions WD40 domain alpha-COP COPI-interacting proteins. Previous work in lab showed that this is required for co-immunoprecipitation with Nucleolin. Because exclusively found cytoplasmic compartment, we hypothesized but previous co-immunoprecipitations had only been performed from whole cell lysates. Alpha-COP has shown bind mRNA, it unclear whether direct or binding an RNA protein (RBP) would act as bridge mRNA alpha-COP. acts RBP both nuclear compartments. We hypothesize some bound are present due their association Nucleolin.
 Experimental Design Project Methods:The first aim project optimize reproducible fractionation protocol reliably separate The second identify co-immunoprecipitated HEK293T cells where can easily express tagged versions Nucleolin-bound previously identified Hela cells. began by testing expression these mRNAs 293-TT using published RT-PCR primers.
 Results:After tests Ftl (Ferritin light polypeptide) highly abundant transcript 293-TTs, immunoprecipitation expressing epitope confirmed complex mRNA.
 Potential Impact:Future experiments will use short-hairpin knockdown determine levels reduced absence