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Rational And Hypothesis : Factors That Affect The Rate Of Mutant Upf1 On Rna B

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Rational and Hypothesis: My hypothesis in this aim is that mutations in UPF1 inhibit NMD thereby stabilizing premature transcripts that are translated to produce truncated proteins which promote pancreatic ASC. Previous studies have demonstrated that a subset of transcripts inhibited upon UPF1 knockdown are stabilized and translated. My objectives in this aim are: a) To determine global occupancy of mutant UPF1 on RNA b) To analyze if the mutant UPF1 bound RNA fragments are translated c) To determine which of the targets are essential to promote tumorigenesis.
A. To identify direct targets of UPF1 mutants in pancreatic cancer.
One challenge facing study of UPF1 and mammalian NMD is identification of direct regulatory targets. While genome
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Briefly, cells will be treated with cyclohexamide to arrest translating ribosomes. Extracts from these cells will be treated with RNase I to degrade regions of mRNAs not protected by ribosomes. The resulting 80S monosomes, which contain a ∼30-nucleotide RPF, will be purified on sucrose gradients and then treated to release the RPFs, which are then processed for Illumina high-throughput sequencing. In parallel, poly (A)-selected mRNA from each sample was randomly fragmented, and the resulting mRNA fragments will be processed for sequencing (mRNA-Seq) using the same protocol as that used for the RPFs. In summary, these experiments will pinpoint the targets, which are translated in UPF1 mutant cells.
C. CRISPR depletion screen to identify bonafide NMD targets
In this sub aim, my goal is to identify the fraction of NMD targets that are essential for UPF1 mediated tumorigenesis. For this purpose, I will collaborate with Dr. Vidigal who has developed a rapid cloning method for generating double guide-paired library and a computational tool for selecting guides that have minimum off-targets. Based on results from Aim 1 and 2a, I will design a customized library targeting UPF1 bound RNA targets. I will infect the library at low multiplicity of infection (MOI) into HPNE KRAS UPF1 mutant cells. I will culture cells for 30 days post transduction to maximize the identification of UPF1 mutant targets
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