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.