Caenorhabditis elegans SMG-2 selectively marks mRNAs containing premature translation termination codons.
Eukaryotic mRNAs containing premature translation termination codons (PTCs) are rapidly degraded by a process termed "nonsense-mediated mRNA decay" (NMD). We examined protein-protein and protein-RNA interactions among Caenorhabditis elegans proteins required for NMD. SMG-2, SMG-3, and SMG-4 are orthologs of yeast (Saccharomyces cerevisiae) and mammalian Upf1, Upf2, and Upf3, respectively. A combination of immunoprecipitation and yeast two-hybrid experiments indicated that SMG-2 interacts with SMG-3, SMG-3 interacts with SMG-4, and SMG-2 interacts indirectly with SMG-4 via shared interactions with SMG-3. Such interactions are similar to those observed in yeast and mammalian cells. SMG-2-SMG-3-SMG-4 interactions require neither SMG-2 phosphorylation, which is abolished in smg-1 mutants, nor SMG-2 dephosphorylation, which is reduced or eliminated in smg-5 mutants. SMG-2 preferentially associates with PTC-containing mRNAs. We monitored the association of SMG-2, SMG-3, and SMG-4 with mRNAs of five endogenous genes whose mRNAs are alternatively spliced to either contain or not contain PTCs. SMG-2 associates with both PTC-free and PTC-containing mRNPs, but it strongly and preferentially associates with ("marks") those containing PTCs. SMG-2 marking of PTC-mRNPs is enhanced by SMG-3 and SMG-4, but SMG-3 and SMG-4 are not detectably associated with the same mRNPs. Neither SMG-2 phosphorylation nor dephosphorylation is required for selective association of SMG-2 with PTC-containing mRNPs, indicating that SMG-2 is phosphorylated only after premature terminations have been discriminated from normal terminations. We discuss these observations with regard to the functions of SMG-2 and its phosphorylation during NMD.
Johns
Lisa
L
Department of Genetics, University of Wisconsin, Madison, WI 53706, USA.
Grimson
Andrew
A
Kuchma
Sherry L
SL
Newman
Carrie Loushin
CL
Anderson
Philip
P
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000918:Antibody Specificity; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D018389:Codon, Nonsense / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D029742:Caenorhabditis elegans Proteins; D018389:Codon, Nonsense; C092470:NMD2 protein, S cerevisiae; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C121027:SMG-2 protein, C elegans; D029701:Saccharomyces cerevisiae Proteins; C431142:Smg-4 protein, C elegans; D015534:Trans-Activators