Caenorhabditis elegans SMG-2 selectively marks mRNAs containing premature translation termination codons

Abstract

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.

FIG. 1.

(A) Demonstration of antibody specificity. A Western blot of total protein from 40 adult worms was probed with anti-SMG-2 (α-SMG-2) (panel 1), anti-phospho(Ser/Thr)Gln dipeptides [α-Phospho(S/T)Q] (panel 2), anti-SMG-3 (α-SMG-3) (panel 3), and anti-ACT-1 (α-ACT-1) (panel 4) antibodies (Ab). WT (lane 1), wild-type N2 strain. All smg alleles used are known from this or previous work to express none of the encoded protein. The strain containing smg-7(r1197), which is temperature sensitive despite being a null allele, was grown at 25°C. (B) Demonstration of IP specificity. The IP pellets and the input following IP with the anti-SMG-2 (left), anti-SMG-3 (center), and anti-SMG-4 (right) antibodies from crude WT and smg-2(−), smg-3(−), and smg-4(−) protein extracts were probed by Western blotting for the presence of SMG-2, SMG-3, and DYN-1 (as a negative control).

FIG. 2.

Interactions of SMG-2, SMG-3, and SMG-4. (A) Proteins contained in crude extracts and in the pellets following IP of SMG-2 were electrophoresed and probed on Western blots with an anti-SMG-3 antibody. Blots were then stripped and reprobed with an anti-SMG-2 antibody. The input was also probed with an antiactin antibody as a loading control. (B and C) As in panel A, except IPs were performed with an anti-SMG-3 antibody (B) or an anti-SMG-4 antibody (C). Western blots were probed first for SMG-2 and subsequently for SMG-3.

FIG. 3.

SMG-2 preferentially associates with PTC-containing rpl-12, rpl-10a, and rsp-4 mRNA. (A) Schematic diagram of rpl-12 alternative splicing. rpl-10a and rsp-4 are alternatively spliced in a similar manner (49, 50). (B) SMG-2-containing mRNPs were immunoprecipitated from wild type (WT) and smg(−) mutant extracts as described in Materials and Methods, and RNA was extracted from the inputs and IP pellets. The entire IP pellet sample and about 5% of the input sample were then analyzed by RT-PCR. The intensity of each band was quantified from a digital image of the stained gel. Numbers below each lane represent the measured ratio of rpl-12(PTC) to rpl-12(+) band intensities from five (lanes 1 to 3 and 6 and 7) or eight (lanes 4 and 5) independent IP experiments followed by RT-PCR (average ± standard deviation). Measured quantities of mRNAs are shown in the graph below; error bars indicate standard deviations. (C and D) As in panel A, except RT-PCR was performed for rpl-10a or rsp-4. Numbers below each lane represent the measured PTC/+ ratios and standard deviations from 3 (lanes 1 to 3 and 6) and 4 (lanes 4 and 5) independent IP or RT-PCR experiments. ND, not determined.

FIG. 4.

Summary of results for selective association of PTC-containing mRNAs with SMG-2. The PTC/+ ratios of five tested mRNAs were measured in crude extracts and in IP pellets after IP of SMG-2 (Fig. 3). The PTC/+ ratios of the IP pellets divided by the PTC/+ ratios of the input are shown for four different smg mutants and five tested genes. Bar heights represent average ratios of three to eight independent IPs (see Fig. 3). Such ratios indicate the increase (fold) in PTC/+ ratios after IP.

FIG. 5.

SMG-3 and SMG-4 do not associate with rpl-12 mRNA. SMG-3-containing mRNPs or SMG-4-containing mRNPs were immunoprecipitated in three independent experiments and analyzed as described in the legend to Fig. 3. RT-PCR products from the IP pellets were consistently too faint to accurately quantify. In the upper panels, the exposure times of the anti-SMG-3 (IP αSMG-3) and anti-SMG-4 (IP αSMG-4) images are the same as that of the input. In the lower panels, the band intensities in the IP pellets have been digitally enhanced to show that the small quantity of mRNAs present is not enriched for either rpl-12(+) or rpl-12(PTC).