Genetic characterization of smg-8 mutants reveals no role in C. elegans nonsense mediated decay

Abstract

The nonsense mediated decay (NMD) pathway degrades mRNAs bearing premature translation termination codons. In mammals, SMG-8 has been implicated in the NMD pathway, in part by its association with SMG-1 kinase. Here we use four independent assays to show that C. elegans smg-8 is not required to degrade nonsense-containing mRNAs. We examine the genetic requirement for smg-8 to destabilize the endogenous, natural NMD targets produced by alternative splicing of rpl-7a and rpl-12. We test smg-8 for degradation of the endogenous, NMD target generated by unc-54(r293), which lacks a normal polyadenylation site. We probe the effect of smg-8 on the exogenous NMD target produced by myo-3::GFP, which carries a long 3' untranslated region that destabilizes mRNAs. None of these known NMD targets is influenced by smg-8 mutations. In addition, smg-8 animals lack classical Smg mutant phenotypes such as a reduced brood size or abnormal vulva. We conclude that smg-8 is unlikely to encode a component critical for NMD.

Figure 1. smg-8 lacks the vulva phenotype associated with mutations in other NMD genes.

(A) Schematic representation of the tm2937 allele, which contains a 272 bp deletion and a 1 bp insertion. This deletion encompasses 22 bp upstream of the start site and the first two exons. Arrows indicate primers used for RT-qPCR (B) Vulval protrusion is one of the phenotypes of canonical smg genes. Left panel shows a wildtype vulva, middle panel shows a smg-8(tm2937) mutant and right panel shows a smg-1(r861) mutant. smg-8 mutants are similar to wild-type and not to smg-1. Arrowheads denote the vulva.

Figure 2. smg-8 lacks an NMD phenotype for the native NMD target rpl-7a.

(A) Schematic representation of the two alternatively spliced isoforms of rpl-7a. The isoform containing the premature termination codon (PTC) is subject to degradation by NMD, whereas the shorter isoform is not. RT-PCR was performed using a pair of primers that distinguish the two spliced isoforms (purple arrows). (B) The upper, PTC band is visible only when the NMD pathway is compromised by smg-1, smg-2 or smg-3 mutations (lanes 2, 3 and 4). Only the lower WT band is observed in wild-type (lane 1) and smg-8 mutant (lane 5) animals. (C) Wild-type worms were fed bacteria expressing dsRNA targeting smg-1, smg-8 or smg-9 from the Ahringer dsRNA library . RNA was analyzed as in (B). (D) An enhanced RNAi mutant strain eri-6/7 , was used and RNAi conducted as in (C). RNA was analyzed as in (B). (E) As in D, using the smg-8(tm2937) mutant strain. (F) RT-qPCR using primers flanking the PTC-containing isoform of rpl-7a, mRNA levels were calculated using the delta-delta-CT method, relative to the control gene pmp-3 . Fold enrichment of the PTC mRNA was normalized to 1 for wild-type. The smg-1 and smg-3 mutants show an enrichment of 15 and 38 fold, respectively. In contrast, in smg-8 mutants, the accumulation of the PTC containing isoform is similar to wild-type (0.7 fold enrichment). smg-8 and eri-6/7 mutant worms treated with smg-8 RNAi show 0.7 and 0.4 fold enrichment, respectively. (G) RT-qPCR to quantify smg-8 RNA. mRNA levels were calculated using the delta-delta-CT method, relative to the control gene pmp-3 . Fold enrichment was normalized to 1 for wild-type. smg-8 and eri-6/7 worms treated with smg-8 RNAi show 0.3 and 0.26 fold enrichment, respectively. (H) As in (G) for wild-type animals and a negative control that lacked Reverse Transcriptase (No RT). Fold enrichment was normalized to 1 for wild-type. No RT control shows 0.3 fold enrichment.

Figure 3. smg-8 lacks an NMD phenotype for the native NMD target rpl-12.

(A) RT-PCR was performed using a pair of primers that distinguish the two spliced isoforms of rpl-12; the upper, PTC band is visible only when the NMD pathway is compromised by smg-1, smg-2 or smg-3 mutations (lanes 2, 3 and 4). Only the lower, WT band is observed in wild-type (lane 1) and smg-8 mutant (lane 5) animals. (B) Wild-type worms were fed bacteria expressing dsRNA targeting smg-1, smg-8 or smg-9 from the Ahringer dsRNA library . RNA was analyzed as in (A). (C) As in (B), using an enhanced RNAi mutant eri-6/7 , . (D) As in (B), using the smg-8(tm2937) mutant strain.

Figure 4. smg-8 is not required for endogenous NMD in C. elegans.

(A) unc-54 gene schematic. The r293 allele contains a 256 bp deletion within unc-54 that includes the 3′ cleavage and polyadenylation site. (B) smg-8(tm2937) or smg-1(r861) mutations were combined with unc-54(r293). Two worms were placed in the middle of the bacterial lawn and allowed to crawl for 45 minutes. Wild-type worms that crawl (top left) leave tracks in the lawn whereas unc-54(r293) mutants cannot move well (top right). smg-1(r861) suppresses unc-54(r293) mRNA degradation and restores movement (bottom left). In contrast, smg-8 does not suppress the paralysis phenotype (bottom right). Arrowheads indicate the tracks left by paralyzed worms.

Figure 5. smg-8 does not restore expression of myo-3::GFP, an exogenous NMD target.

(A) Schematic representation of the exogenous NMD GFP reporter, driven by the myo-3 promoter, which is destabilized by an amino acid sequence that marks a protein for degradation (degron), and a long 3′UTR . (B) smg-8 and control mutations were introduced into CL724 (myo-3::GFP) worms. The double combinations were then inspected under a fluorescent microscope. smg-1 and smg-3 mutants express high levels of GFP. In contrast, smg-8 animals photographed under the same conditions show only a slight accumulation of GFP, similar to the wild type.