To identify how UPF1 mutations alter gene expression, I will utilize CRISPR based genome editing (Doudna and Charpentier 2014) to generate human cell lines that contain UPF1 mutations identified in pancreatic ASC. To examine the differentially expressed candidates in pancreatic ASC, I will isolate total RNA from UPF1 mutant cell lines and perform RNA sequencing. To establish whether the upregulated genes are direct targets of NMD, I will compare this data with RNA isolated from cells where UPF1 is downregulated using short hairpin RNAs. To validate the top targets from RNA sequencing experiments, I will perform qPCR and northern blotting. To examine whether these candidates are also altered in pancreatic ASC, I will analyze the RNA levels of top targets in pancreatic ASC samples that contain UPF1 mutations.
To determine the function of the identified candidates from high-throughput sequencing, I will perform overexpression and knockdown experiments in primary pancreatic cell lines to examine for corresponding gain or loss in proliferation. In addition, I will determine whether overexpression of the candidates transforms primary pancreatic cells. In summary, results from this aim will elucidate the mechanism by which UPF1 mutations promote pancreatic …show more content…
Since Upf1 knockout is embryonic lethal but Upf1 heterozygotes are normal (Medghalchi, Frischmeyer et al. 2001), I will generate conditional Upf1 mutants. Previous studies have shown that majority of UPF1 mutations identified in pancreatic ASC promotes skipping of exons 10 and exon11 that cause a truncation in the essential RNA helicase domain. Mutations in the RNA helicase domain of UPF1 has been shown to be dominant negative(Sun, Perlick et al. 1998). To model this exon-skipping event, I will generate a targeting construct that will place the loxP sites on the introns flanking exon 10 and exon11 upon homologous recombination in ES cells. To control the expression of the floxed Upf1 allele, I will place a lox-stop-lox cassette in the first intron of Upf1 that causes transcription termination in the absence of Cre-recombinase. To restrict expression of Upf1 mutants to the pancreas, I will cross the Upf1 floxed mice with PDX1-CRE expressing mice(Hingorani, Petricoin et al. 2003).
To determine whether Upf1 mutants promote pancreatic ASC, I will monitor 20 wild type controls and 20 mutant animals. Survival curves, tumor incidence, and pancreatic tumor types arising in wild type and mutant animals will be compared. In conclusion, these experiments will demonstrate whether UPF1 mutations are essential for pathogenesis of cancer in mice and potentially in