Nonsense-mediated mRNA decay, or NMD, functions to degrade mRNAs containing premature stop codons and has been conserved throughout the Eukarya . In C. elegans , function of the seven smg genes is required for NMD.
smg-5 and
smg-7 encode proteins of 549 and 459 amino acids, respectively. Both are novel genes, though
smg-7 contains two TPR (tetratricopeptide repeats) domains, which are known in other proteins to mediate protein-protein interactions. We have previously demonstrated by co-immunoprecipitation studies that SMG-5 and SMG-7 are members of a protein complex. Two-hybrid technology was utilized to determine whether these proteins interact directly. A two-hybrid screen using
smg-7 as bait recovered multiple
smg-5 clones. To delineate the interaction domains, we undertook a series of two-hybrid tests using a series of
smg-5 and
smg-7 subclones. SMG-7 requires both TPR domains for an interaction with SMG-5. The amino terminal third of SMG-5 is not required for an interaction with SMG-7. Further mapping of interaction domains is in progress. We have discovered a probable cycle of SMG-2 phosphorylation / dephosphorylation that is important for NMD. In
smg-5(-) ,
smg-6(-) or
smg-7(-) mutants, a phosphorylated isoform of SMG-2 accumulates to abnormally high levels. This accumulation is dependent on the wild-type function of
smg-1 ,
smg-3 and
smg-4 . For example, a
smg-1(-)
smg-5(-) double mutant does not accumulate phosphorylated SMG-2. Significantly,
smg-1 encodes a probable protein kinase, although we do not yet know whether SMG-1 is the direct kinase of SMG-2. The smg genes may be classified into two groups, those required to phosphorylate SMG-2 and those that ensure no phosphorylated SMG-2 accumulates. We have surveyed
smg-2 alleles for those that may be defective in dephosphorylation / phosphorylation. Of seven
smg-2(-) alleles that encode full length protein, two accumulate phosphorylated SMG-2. These alleles may encode proteins that are incapable of dephosphorylation. We believe the genetic evidence argues that phosphorylation and dephosphorylation of SMG-2 is central to NMD.