| Messenger RNAs containing premature translation termination codons (PTCs) are identified and targeted for degradation by nonsense-mediated mRNA Decay (NMD). NMD requires the function of at least seven genes, smg-1 through smg-7, in C. elegans and humans. I used immunoprecipitation experiments to assess the formation of SMG-2-containing complexes in numerous smg(-) mutants. I discovered the following: (i) SMG-2 interacts with SMG-3 and SMG-4, and this complex is not influenced by the phosphorylation state of SMG2. (ii) SMG-2 preferentially associates with PTC-containing mRNAs. I interpret this to indicate that, when PTCs are identified, SMG-2 marks the mRNAs as PTC-containing. SMG2 marking of PTC-containing mRNAs is dependent on SMG-3, SMG-4, and the helicase domain of SMG-2. This suggests that SMG-3, SMG-4, and the SMG-2 helicase domain are either involved in PTC identification or in the subsequent mRNP remodeling that results in SMG-2 marking of PTC-containing mRNAs. SMG-2 marking of PTC-containing mRNAs occurs independently of SMG-1 and SMG-5, indicating that the cycle of SMG-2 phosphorylation is required at a step after PTC-marking. (iii) Neither SMG-3 nor SMG-4 are detectably associated with SMG-2-marked, PTC-containing mRNAs, suggesting that they exit the mRNP after marking. (iv) Independent missense alleles of smg-2 disrupt NMD either upstream or downstream of marking, indicating that functions of SMG-2 are required throughout NMD. (v) Epistasis analysis demonstrate that smg-3 and smg-4 function upstream of smg-1 and smg-5, and that the helicase domain of SMG-2 functions upstream of smg-1 in the pathway of NMD. (vi) Finally, the PTC-containing mRNAs associated with and marked by SMG-2 are capped. Therefore, if decapping is a degradation step in the decay of PTC mRNAs in C. elegans, decay itself has not yet been initiated on SMG-2-marked mRNAs. |
