Worm Breeder's Gazette 15(2): 43 (February 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Laboratory of Genetics, University of Wisconsin-Madison, Madison WI 53706
Nonsense-mediated mRNA decay is a surveillance system that rapidly degrades messenger RNAs containing premature stop codons. Nonsense-mediated mRNA decay has been observed in many eukaryotes including yeast, Drosophila, humans and plants. This nonessential system of decay has been characterized genetically in C.elegans. Function of the seven smg genes is required for nonsense-mediated mRNA decay. Mutations in any of these genes abolish NMD, and nonsense mutant mRNAs accumulate to wild-type levels. smg-2 encodes a protein containing a probable C6 zinc finger domain, and a type I RNA helicase domain with a nucleotide-binding motif. We raised antibodies against the amino terminal portion of a His-tagged SMG-2, fusion protein. These antibodies detect, on a western blot, a single band of the expected 120 Kd in N2 animals. No signal is detected in smg-2 null mutants. The same single 120Kd band is found in smg-1, smg-3 and smg-4 mutants. In smg-5, smg-6 and smg-7 mutants, however, a more slowly migrating SMG-2 isoform is present in addition to the abundant 120Kd isoform. When smg-5 extracts are treated with lambda phosphatase, the more slowly migrating isoform is no longer detected, demonstrating that SMG-2 is a phosphoprotein and that the phosphorylated form accumulates in smg-5, smg-6 and smg-7 mutants. We believe that phosphorylated SMG-2 is too transient to detect in N2 worms. We have tested which smg genes are required for SMG-2 phosphorylation by double-mutant analysis. A smg mutant that accumulates the phosphorylated form of SMG-2 can be combined with a second smg mutation. If no phosphorylated form is detected in the double mutant, the second smg gene is required for SMG-2 phosphorylation. From this double mutant analysis, smg-1, smg-3 and smg-4 are required for phosphorylation of SMG-2. SMG-1 encodes a putative PI-3/protein kinase domain. We are currently testing whether SMG-1 interacts with SMG-2 and if a smg-1-dependent kinase activity coimmunoprecipates with SMG-2. The analysis of SMG-2 in smg single and double mutants divides the smg genes into two functional classes, those required to phosphorylate SMG-2 and those required for normal dephosphorylation of SMG-2. This is the first grouping of genes involved in NMD and it is hoped this will provide a framework on which to build a biochemical pathway.