Maddy Ford

Altering Conserved Residues on Dbp5 in S. cerevisiae to Assess Function

Nuclear mRNA export is essential for the viability of eukaryotic cells as it enables gene expression. This process allows for mRNA, the genetic copy of DNA that encodes for proteins, to be transported from the nucleus, where it is generated, to the cytoplasm for translation. The mRNA transcript requires several binding proteins to cross through nuclear pore complexes (NPC), which are selective doorways embedded in the nuclear envelope1. Mex67 is a protein that binds to mRNA, allowing for the transcript to be transported across the nuclear envelope. Once the transcript is in the cytoplasm, another protein called Dbp5 binds to the mRNA and detaches Mex67 to prevent the mRNA from re-entering the nucleus2. Along with Mex67, several other proteins bind to mRNA in the nucleus and remain on the transcript in the cytoplasm. How Dbp5 selectively removes Mex67, and not other RNA binding partners is unknown. Dbp5 has several binding partners, but a patch of conserved amino acids on Dbp5 has previously been identified that is not known to be at interface with known binding partners. This patch having been evolutionarily maintained, indicates that the site likely holds a significant function for cells. I hypothesize that this patch on Dbp5 are at interface with Mex67. In order to test the hypothesis, mutations to the codons encoding the conserved amino acids lysine 117 and glutamic acid 120 were created in order to test the functionality of the site using site-directed PCR mutagenesis. The mutated plasmids were then transformed into E.coli to be amplified, sequenced to confirm the mutations, and then were transformed into S. cerevisiae to assess the mutant Dbp5 functionality. If this patch of conserved amino acids is essential for mRNA export, the Dbp5 mutants will result in non-functional cells. Due to the wild-type Dbp5 having no growth on 5-FOA plates, I am unable to conclude the function of the Dbp5 mutants and if the amino acids lysine 117 and glutamic acid 120 are essential for the viability of eukaryotic cells.

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