It has been found that over 30% of the mRNA transcripts generated by alternative splicing contain a premature termination codon, which makes them possible targets for nonsense mediated mRNA decay (NMD). There is no comprehensive list of mRNA isoforms that are degraded by NMD, and transcription rates of NMD targeted transcripts are unknown. How does coupled alternative splicing and NMD regulate the level of protein produced?

We will inhibit the machinery of NMD in human and mouse cell lines and then identify which alternatively spliced mRNA isoforms are subject to NMD using a splice junction specific microarray. We will use RNAi to knock down the expression of Upf1, inhibiting NMD by removing one of the essential components of the NMD machinery. Western blots will be used to verify that Upf1 protein levels have decreased, and RT-PCR will verify that NMD is inhibited.

Once we have proven that NMD has been inhibited by our methods, we will prepare the mRNA from NMD-inhibited and mock-transfected cells to be used in microarray experiments. We will be manufacturing our own splice junction specific microarrays to probe human genes, and we will be using the Affymetrix alternative splicing chip to probe mouse genes. The results will allow us to generate a list of mRNA isoforms that are degraded by NMD and allow us to get an idea of the transcription rates of NMD targeted transcripts. From this starting point we can begin to decipher how alternative splicing and NMD work together to regulate protein levels.

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Previous work done by Georgeann O'Brien : IDENTIFYING AND VALIDATING TISSUE-SPECIFIC ALTERNATIVE SPLICING EVENTS IN MOUSE BRAIN GENES