Genome wide analysis of a novel human mRNA export pathway. (360G-Wellcome-083130_Z_07_Z)

We have identified a novel mRNA export factor, UIF likely to define a new mRNA export pathway in human cells. UIF associates with mRNA export factors UAP56 and TAP and can promote export of an otherwise inefficiently transported mRNA. Mass spectrometry reveals that UIF copurifies with the SSRP1 component of the FACT chromatin remodeling complex. Here we will carry out a genome wide analysis of the dependency of mRNAs on UIF, ALY, DDX39+UAP56 and the FACT complex for export. We will define the mR NA substrates for the new UIF pathway using immunoprecipitation of UIF, extraction of bound mRNA followed by microarray analysis (RIP-CHIP). Secondly we will knockdown UIF, ALY/REF, UAP56 together with the paralogue DDX39 and the SSRP1 and SPT16 components of FACT. Total and cytoplsmic RNA will be analysed using microarrays to determine which pathways individual mRNAs use during export. We will characterize the RNA binding properties of UIF using cross-linking immunoprecipitation (CLIP) to dete rmine where it binds on mRNAs and use deletions and mutations to define the RNA binding domain. We will also examine whether UIF has the ability to switch TAP from a low to high affinity RNA binding state.