The steady-state level of a messenger RNA is established by its relative rates of synthesis and degradation. It is increasingly apparent that mRNA degradation is an important aspect of gene expression and its regulation. Although numerous cis-acting sequences that regulate mRNA stability have been defined, little is known about trans-acting factors that selectively degrade a message. Most mRNA turnover appears to be intimately coupled to translation. This is particular clear in the case of nonsense mutant mRNAs. mRNAs that contain nonsense mutations (premature stop codons) are unstable in all organisms, including humans. We will study this phenomenon, termed nonsense-mediated mRNA decay, in the nematode Caenorhabditis elegans. Loss-of-function mutations affecting any of six different genes (smg-1 through smg-6) eliminate the C. elegans system of nonsense-mediated mRNA decay. The six smg genes define proteins that unambiguously act in trans to accomplish specific mRNA turnover. We will study genetic and molecular properties of the smg genes and answer the following questions: What are the smg gene products? Where are smg proteins located in the cell? With what do they associate? And, how are nonsense mutant mRNAs targeted for degradation? Our methods combine genetic, molecular, and biochemical approaches. Specific smg genes will be cloned and sequenced. smg genes essential for viability of the animal will be identified using a novel genetic screen. Polyclonal anti-Smg antibodies will be used to locate smg proteins in situ and in fractionated cell extracts. Site-directed mutations that test specific models for the specificity of nonsense-mediated decay will be constructed. Our results should identify the proteins involved in nonsense-mediated decay and provide an outline understanding of how the system selects mRNAs to be destroyed. Our long range goals are to understand the molecular mechanisms of mRNA turnover. Nonsense-mediated decay in C. elegans may prove to be similar to many cases of constitutive or regulated mRNA turnover. Nonsense-mediated mRNA decay is a universal phenomenon and may contribute to human genetic disease. Patients having nonsense alleles of an affected gene usually express low steady-state levels of the mutant mRNA. We believe that these and other mRNAs are degraded by the human system of nonsense-mediated mRNA decay. Understanding the smg genes of C. elegans will contribute to understanding the etiology of these diseases.
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