Nonsense-mediated decay (NMD) is a quality-control mechanism that degrades aberrant mRNAs harboring premature termination (nonsense) codons (PTCs). One-third of human genetic diseases are caused by PTC- containing mutant genes with nonsense or frameshift mutations. Recently, it has become apparent that NMD also acts on a subset of wild-type genes. Current estimates from microarray analyses from several groups indicate that between 5 and 10% of the genes in Saccharomyces cerevisiae, Drosophila melanogaster, and mammals give rise to transcripts that are subject to NMD. The ability of NMD to modulate the level of normal transcripts leads to the hypothesis that NMD is itself subject to regulation. Consistent with this hypothesis, there is evidence that the magnitude of NMD varies in different cell types and at different developmental stages. However, the molecules and pathways that modulate NMD have not been elucidated. This proposal revolves around 2 recent discoveries indicating that NMD is under negative regulatory control. First, a neuron-specific microRNA (miRNA) was discovered that negatively regulates 3 factors that have a role in NMD: the RNA-helicase UPF1, the exon-junction complex (EJC) core component MLN51, and the ribonuclease DCP2. This suggests that this miRNA regulates NMD itself, which is supported by preliminary data. This miRNA is expressed during both embryonic and postnatal development of the brain;in the adult, it is most highly expressed in the hippocampus and olfactory lobe. This application addresses the role of this miRNA in neuronal development and function. Experiments are also proposed to determine the mechanisms by which this miRNA controls gene expression through its targets UPF1, MLN51, and DCP2. Finally, the full spectrum of mRNAs regulated by this miRNA will be investigated. The second example of negative regulatory control is that the EJC-associated factor UPF3A is downregulated by its paralog UPF3B. This is a conserved downregulatory response that is disrupted in humans with UPF3B mutations (whose phenotype is mental retardation). The UPF3A gene may have been selected for during the evolution of higher eukaryotes because its paralog, UPF3B, is present on the X chromosome, which is known to be transcriptionally inactivated during meiosis in male germ cells. In this proposal, the function of UPF3A in male germ cells and other cell types is addressed. Also addressed is why UPF3A expression is specifically suppressed in cell types that have UPF3B. Finally, the molecular mechanism of UPF3A suppression and relief from this suppression will be examined. The broad goals of this proposal are to elucidate the underlying mechanism for these negative regulatory circuits and to begin to understand the physiological consequences of this regulation. By elucidating how NMD is regulated, a foundation will be made for elucidating the NMD transcriptional networks in cells, which in turn may lead to therapies for diseases involving the NMD RNA surveillance pathway.
Nonsense-mediated decay (NMD) is a quality-control pathway that protects cells from genetic mistakes. The genetic mistakes detected by NMD (those that generate premature termination codons) are responsible for one-third of human genetic diseases. This proposal addresses newly discovered mechanisms responsible for regulating NMD that are particularly relevant for understanding normal neurological function and the genetic basis for mental retardation.
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