Cells have evolved quality control mechanisms to identify, remove, and mitigate harm from abnormal gene expression and its intermediates. Among these mechanisms is Nonsense-Mediated mRNA Decay (NMD). During NMD, cells identify and then degrade mRNAs with premature stop codons encoding truncated, deleterious, and potentially dominant-acting proteins. NMD is intimately related with human disease: ~11% of all human inherited diseases are caused by premature stop codons. Despite much effort, an understanding of how NMD target mRNAs are identified and then removed remains elusive. Our ?long-term goal? is to illuminate how cells recognize and repress mRNAs via NMD in metazoa (animals). The ?key focus? of this proposal is the second step of this process: the molecular details of how NMD targets are removed from the cell. My lab employs the animal ?C. elegans?, in which the NMD machinery is non-essential, allowing us to exploit powerful genetic approaches not readily available in other animals. Two factors key for NMD target mRNA clearance are the RNA helicase SKI and the ribosome rescue factor PELO. When cells lack SKI and PELO, ribosomes stall on truncated premature stop codons of NMD target mRNAs. We demonstrated that ribosomes stalled on truncated stop codons represent an intermediate of NMD, providing a novel functional landmark in the process of mRNA removal by NMD. Building on this observation, our specific aims are:
(Aim 1) We will characterize the nature and timing of RNA cleavage event(s) that give rise to ribosomes stalled on NMD target mRNAs. We will use reporters designed to test distinct models of the spatio-temporal relationship between RNA cleavage and premature translation termination.
(Aim 2) We will illuminate the molecular mechanisms by which SKI and PELO remove RNAs and ribosomes during NMD, leveraging our facile ability to mutate SKI and PELO, and to assay their function ?in vivo?.
(Aim 3) Finally, we will address a question fundamental to the perceived role of NMD in preventing truncated protein production: How does NMD affect protein production from its target mRNAs? We will determine what protein species are produced during NMD, and determine whether there is repression of nascent protein chains as NMD target mRNAs are degraded. We expect that a clear picture of the cascade of events that comprise target mRNA clearance during NMD will (1) inform models of preceding events, ?i.e.?, how premature stop codons are recognized; (2) illuminate the molecular events of co-translational mRNA decay, important for understanding normal gene expression and regulation; and (3) yield a clear picture of what mRNAs and proteins are expressed from genes harboring premature stop codons, with mechanistic implications for NMD?s roles in human diseases.
This proposal investigates the steps by which cells identify and remove mRNAs encoding prematurely terminated proteins, using a powerful combination of genetic, genomic, and biochemical approaches. By determining the intermediates and products of this process, we will (1) illuminate molecular mechanisms of mRNA removal upon premature translation termination, and (2) yield insight into a diverse number of human genetic diseases caused by mutations that yield premature translation termination.
