Identification of Dbp5-Nab2 Binding Interface using a Split-Venus Reporter Screen

In eukaryotic cells, mRNA is the genetic information that has the ability to exit the nucleus in order for the information to be translated into functional proteins. Three proteins, Mex67, Nab2, and Mtr2, play an essential role in directing the mRNA out of the nucleus through nuclear pore complexes, doorways in the nuclear envelope. Once the mRNA reaches the cytoplasm, the protein DBP5 removes these three proteins off of the mRNA so they can be recycled back into the nucleus to allow for additional transcript export. In addition to these proteins, the mRNA is coated with other proteins that need to remain on the transcript for translation and other cytoplasmic processes. It is unknown how DBP5 specifically removes Mex67, Nab2, and Mtr2 from mRNA and not these other proteins. We hypothesize that DBP5 directly interacts with one of the removed proteins while they are associated with RNA to then induce their release from the transcript. Indeed, previous experiments have demonstrated a specific interaction between Dbp5 and Nab2 via a split-Venus experiment. However, in this experiment, Dbp5 and Nab2 can become stably bound together, prohibiting recycling of Nab2 and cause inviability. To test our hypothesis, we took advantage of this result, to perform a random mutagenesis screen within Dbp5 to identify mutants that disrupt interaction with Nab2. We reasoned that these mutants would aid in identification of the putative Dbp5-Nab2 interface. Error-prone PCR mutagenesis was performed during DBP5 ORF amplification, and the resulting amplicon library was cloned into an overexpression split-Venus plasmid. We reasoned that if the mutation disrupts the Dbp5-Nab2 interface, the two will not interact, will not then be locked together, and thus the cells with this result will be viable. From this experiment, the plasmids from viable cells will be re-isolated from yeast and sequenced. If successful, the mutations resulting in a disruption of interactions will be further analyzed. We anticipate that this study will begin to shed light on how dynamic RNA-binding protein interactions are regulated during gene expression