SMG-1 is a phosphatidylinositol kinase-related protein kinase required for nonsense-mediated mRNA Decay in Caenorhabditis elegans

Abstract

Eukaryotic messenger RNAs containing premature stop codons are selectively and rapidly degraded, a phenomenon termed nonsense-mediated mRNA decay (NMD). Previous studies with both Caenohabditis elegans and mammalian cells indicate that SMG-2/human UPF1, a central regulator of NMD, is phosphorylated in an SMG-1-dependent manner. We report here that smg-1, which is required for NMD in C. elegans, encodes a protein kinase of the phosphatidylinositol kinase superfamily of protein kinases. We identify null alleles of smg-1 and demonstrate that SMG-1 kinase activity is required in vivo for NMD and in vitro for SMG-2 phosphorylation. SMG-1 and SMG-2 coimmunoprecipitate from crude extracts, and this interaction is maintained in smg-3 and smg-4 mutants, both of which are required for SMG-2 phosphorylation in vivo and in vitro. SMG-2 is located diffusely through the cytoplasm, and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation. We discuss the role of SMG-2 phosphorylation in NMD.

FIG. 1.

(A) Western blot of total protein from the wild type (lane 1) and three independent smg-1(−) mutants (lanes 2 to 4) probed with anti-SMG-1 (αSMG-1) antibody (top) and anti-PR65 (αPR65) antibody (bottom) as a loading control. (B) Domain structure of SMG-1. SMG-1 contains a FAT domain (pfam02259), a FATC domain (pfam02260), a PIK domain (pfam00454), and a putative nuclear localization signal (NLS?) at the indicated amino acid positions.

FIG. 2.

Phylogenetic analysis of SMG-1 and SMG-2. (A) Neighbor-joining tree of PIK-related protein kinases. (B) Neighbor-joining tree of the UPF1/SMG-2 subfamily of type I helicases. For both trees, confidence values are shown to the left of selected nodes marked with circles: percent bootstrap values by neighbor joining (top) and percent reliability values by quartet puzzling (bottom) are shown. Supported nodes (i.e., bootstrap values of >70%) indicate that sequences to the right of that node are more closely related to each other than any of them are to any other sequences included in the analysis.

FIG. 3.

The kinase domain of SMG-1 is required for NMD. (A) Western blots of total protein from the wild type (WT) (lane 1), smg-1(r861) (lane 2), and smg-1(r861) expressing either SMG-1(+) or SMG-1(D1966A) (lanes 3 and 4) probed with anti-SMG-1 (αSMG-1) (upper panel) or anti-PR65 (αPR65) antibodies (lower panel) as a loading control. (B) rpl-12 reverse transcription (RT)-PCR assay of the same strains analyzed in panel A. Alternative splicing of rpl-12 pre-mRNA is illustrated, with exons 2 and 3 indicated as numbered boxes. Intron 2 is alternatively spliced to yield in-frame mRNA unaffected by NMD (lower mRNA) and a PTC-containing mRNA that is degraded by NMD (upper mRNA).

FIG. 4.

Phosphorylation of SMG-2 in vitro requires SMG-1 and is sensitive to wortmannin. (A) SMG-2 immunoprecipitation kinase assays. smg-1(r861), smg-2(r908), and smg-5(r860) are null alleles of the respective genes. (B) SMG-2 immunoprecipitation kinase assays in the presence (lanes 4 to 6) or absence (lanes 1 to 3) of 1 μM wortmannin.

FIG. 5.

SMG-1 interacts with SMG-2. (A) Proteins immunoprecipitated (IP) by anti-SMG-2 (αSMG-2) antibodies were electrophoresed and probed on Western blots with anti-SMG-1 (αSMG-1) antibodies. WT, wild type. (B) Western blots of strains used in Fig. 5A probed with anti-SMG-1. (C) Proteins immunoprecipitated by anti-SMG-1 antibodies were electrophoresed and probed on Western blots with anti-SMG-2 antibodies. (D) Proteins immunoprecipitated by anti-SMG-2 antibodies were electrophoresed and probed on Western blots with anti-SMG-1 antibodies. SMG-1(+) and SMG-1(D1966A) are expressed from transgenes in the strains of lanes 4 and 5, respectively. smg-1(r861), smg-2(r908), smg-3(r930), smg-4(r1169), and smg-5(r860) are null alleles of the respective genes.

FIG. 6.

Double labeling of wild-type and smg(−) mutant worm gonads with anti-SMG-2 antibody (red signal) and Mab414, an antibody that stains the nuclear envelope (green signal). Genotypes are wild type (A), smg-2(r908) (B), smg-1(r904) (C), smg-3(r930) (D), smg-4(r1169) (E), smg-5(r860) (F), and smg-2(r866) (G). With the exception of smg-2(r866), all smg alleles are null.