SMG-5, required for C.elegans nonsense-mediated mRNA decay, associates with SMG-2 and protein phosphatase 2A

Abstract

mRNAs that contain premature stop codons are degraded selectively and rapidly in eukaryotes, a phenomenon termed 'nonsense-mediated mRNA decay' (NMD). We report here molecular analysis of smg-5, which encodes a novel protein required for NMD in Caenorhabditis elegans. Using a combination of immunoprecipitation and yeast two-hybrid assays, we identified a series of protein-protein interactions involving SMG-5. SMG-5 interacts with at least four proteins: (i) SMG-7, a previously identified protein required for NMD; (ii) SMG-2, a phosphorylated protein required for NMD in worms, yeasts and mammals; (iii) PR65, the structural subunit of protein phosphatase 2A (PP2A); and (iv) PP2A(C), the catalytic subunit of PP2A. Previous work demonstrated that both SMG-5 and SMG-7 are required for efficient dephosphorylation of SMG-2. Our results suggest that PP2A is the SMG-2 phosphatase, and the role of SMG-5 is to direct PP2A to its SMG-2 substrate. We discuss cycles of SMG-2 phosphorylation and their roles in NMD.

Fig. 1. The domain structure of SMG-5. SMG-5 contains a PINc and two tetratricopeptide (TPR) repeats. smg-5(r860), smg-5(r869) and smg-5(r916) are nonsense alleles.

Fig. 2. Expression of SMG-5 in wild-type and smg-5 mutants. SMG-5 was concentrated from wild-type and mutant extracts by immunoprecipitation and then analyzed by western blotting. Lane 1, purified recombinant His₆-tagged SMG-5; lane 2, wild-type C.elegans; lanes 3–9, seven independent smg-5 alleles. Six tested alleles express no detectable SMG-5 even after prolonged exposure. Smg-5(r916) expresses an ∼42 kDa truncated protein [SMG-5(Q401X)]. Rabbit immunoglobulin (Ig) is present from the immunoprecipitations and detected in these experiments.

Fig. 3. Co-immunoprecipitation of SMG-5 and SMG-2. (A) Total soluble proteins (lanes 3–5) or proteins found in the pellet following immunoprecipitation of soluble extracts with anti-SMG-5 antibody (lanes 1 and 2) were electrophoresed and probed in western blots with anti-SMG-2 antibody. (B) Proteins immunoprecipitated by anti-SMG-7 or anti-SMG-5 antibodies were electrophoresed and probed in western blots with anti-SMG-5 antibodies. (C) Immunoprecipitations and western blotting performed as in (A). Mutants used for this analysis were smg-1(r861), smg-2(r908), smg-3(r867), smg-4(ma116), smg-5(r869), smg-6(r896) and smg-7(r1131).

Fig. 4. Two-hybrid interactions between SMG-5 and SMG-7. ‘His phenotype’ (column 4) refers to the ability (‘+’) or inability (‘–’) of strains to grow on medium lacking histidine. Columns 5 and 6 show β-galactosidase colorimetric plate tests and enzymatic assays, respectively. Standard deviations were calculated from at least three independent assays. LexA DNA-binding domain fusions (‘bait’) and Gal4 activation domain fusions (‘prey’) contained SMG-5 amino acids 1–549 (full length) and 1–546, respectively. All SMG-7 fusion proteins contained SMG-7 amino acids 7–454 (out of 458).

Fig. 5. Two-hybrid interactions tested among SMG proteins and PP2A subunits. Growth tests were performed on histidine-free medium containing 1 mM 3-amino-1,2,4-triazole. ‘Bait’ and ‘prey’ proteins were fused to LexA DNA-binding domains and Gal4 activation domains, respectively.

Fig. 6. SMG-5 and SMG-2 interact with PR65 and PP2A_C. (A–C) Proteins immunoprecipitated by anti-SMG-5 antibody were electrophoresed and probed in western blots with antibodies to PR65, PP2A_C and SMG-2, respectively. (D) Proteins immunoprecipitated by anti-PR65 antibody were probed with anti-SMG-2 antibody. Smg-1(r861), smg-2(r908) and smg-5(r860) are null alleles of the affected genes.