Nonsense-mediated mRNA decay (NMD) is a surveillance system that recognizes transcripts with premature termination codons (PTCs) and degrades them. An estimated 30% of human disease mutations produce PTCs, and NMD is thought to underlie the phenotypes of many of these diseases. Surprisingly, a third of human reliably-inferred natural alternatively-spliced mRNA isoforms also contain PTCs, and are therefore expected to be eliminated by NMD rather than produce protein. This process seems wasteful, like coupling a printer to a shredder, and it may largely represent the production of mis-spliced mRNAs that are cleared by the cell. However, the coupling of alternative splicing and NMD is also known in several instances to have a specific regulatory function, in which a particular alternative splicing event effectively turns off protein expression. We term this regulated unproductive splicing and translation (RUST). We propose to discover the prevalence, functional impact, and evolutionary significance of NMD induced by alternative splicing. Our approach combines both experimental and computational methods.
Our specific aims to accomplish this goal are the following. 1.) We will survey the prevalence of natural mRNAs with PTCs. Computationally, we will analyze EST and other alternative splicing data in all organisms with known PTC-recognition mechanisms. Experimentally, we will use Affymetrix human all-exon DNA arrays to globally survey the prevalence of mRNA isoforms degraded by NMD. 2.) We will attempt to recognize functional instances of RUST by looking for evolutionary conservation, signature sequences, and overlooked experimental evidence in the literature. We expect that these will illuminate our understanding of human biology and disease. We will verify our functional predictions of NMD activity with detailed experimental studies. 3.) We will seek to better understand the evolutionary histories of alternative splicing and NMD, as well as the functional and evolutionary links between them. These studies will also be used to offer insight into the varied mechanisms of PTC-recognition in different species.
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