Mechanistic insights and identification of two novel factors in the C. elegans NMD pathway

Abstract

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs). Seven genes (smg-1-7, for suppressor with morphological effect on genitalia) that are essential for NMD were originally identified in the nematode Caenorhabditis elegans, and orthologs of these genes have been found in several species. Whereas in humans NMD is linked to splicing, PTC definition occurs independently of exon boundaries in Drosophila. Here, we have conducted an analysis of the cis-acting sequences and trans-acting factors that are required for NMD in C. elegans. We show that a PTC codon is defined independently of introns in C. elegans and, consequently, components of the exon junction complex (EJC) are dispensable for NMD. We also show a distance-dependent effect, whereby PTCs that are closer to the 3' end of the mRNA are less sensitive to NMD. We also provide evidence for the existence of previously unidentified components of the NMD pathway that, unlike known smg genes, are essential for viability in C. elegans. A genome-wide RNA interference (RNAi) screen resulted in the identification of two such novel NMD genes, which are essential for proper embryonic development, and as such represent a new class of essential NMD genes in C. elegans that we have termed smgl (for smg lethal). We show that the encoded proteins are conserved throughout evolution and are required for NMD in C. elegans and also in human cells.

Figure 1.

A PTCx transgene harboring a nonsense codon is subject to NMD in C. elegans. (A) Schematic representation of GFP/LacZ reporters. Black boxes represent GFP exons, white boxes represent LacZ exons, and gray boxes represent the 3′UTR. Intervening black lines correspond to introns. The natural stop codon is indicated by an asterisk. In the PTCx reporter, the position where a nonsense codon was generated by site-directed mutagenesis is indicated. (B, panel i) Transgenic worms carrying a wild-type reporter show ubiquitous GFP expression. (Panel ii) This expression is not affected by the depletion of SMG-2. Introduction of a nonsense codon (PTCx) resulted in lack of GFP expression (panel iii), whereas inactivation of NMD by SMG-2 RNAi led to strong GFP expression (panel iv). (C) The level of the PTCx reporter mRNA was monitored by semiquantitative RT–PCR. In the PTCx strain, the level of reporter mRNA is very low (lane 3); however, the level of transgene mRNA is significantly increased upon depletion of the smg-2 transcript (lane 5).

Figure 2.

The position of the PTC within the transgene influences NMD, demonstrating a polarity effect. (A) Schematic representation of PTCx, PTCa, and PTCb reporters. Black boxes represent GFP exons, white boxes represent LacZ exons, and gray boxes represent the 3′UTR. Intervening black lines correspond to introns. The natural stop codon is indicated by an asterisk. The positions of nonsense codons within the penultimate exon of the LacZ gene are indicated. (B) Transgenic worms carrying the PTCx and PTCa reporter lack GFP expression (panels i,iii, respectively); however, GFP expression is restored upon SMG-2 depletion (panels ii,iv, respectively). Transgenic worms carrying the PTCb reporter show robust GFP expression (panel v), which is not altered by the inactivation of NMD by SMG-2 depletion (panel vi).

Figure 3.

Splicing is not required for PTC recognition in C. elegans. (A) Schematic representation of PTCxΔIN and PTCaΔIN reporters. Black boxes represent GFP exons, white boxes represent LacZ exons, and gray boxes represent the 3′UTR. Intervening black lines correspond to introns. The natural stop codon is indicated by an asterisk. Introns within the LacZ gene were removed, placing the nonsense codons within the context of the last exon. Nonsense mutations are the same as in the PTCx and PTCa reporters, respectively. (B) A transgene with no introns downstream from the PTC is subject to NMD. Transgenic worms carrying the PTCxΔIN reporter lack GFP expression (panel i); however, GFP expression is restored upon the inactivation of NMD by SMG-2 depletion (panel ii). Similarly, transgenic worms carrying the PTCaΔIN reporter lack GFP expression (panel iii), which is restored upon the inactivation of NMD by SMG-2 depletion (panel iv). (C) The level of PTCxΔIN reporter mRNA was monitored by semiquantitative RT–PCR. In the PTCxΔIN strain, the level of reporter mRNA is very low (lane 3); however, it is significantly increased upon depletion of SMG-2 (lane 5).

Figure 4.

EJC components and SR proteins are dispensable for NMD in C. elegans. (A) Microinjection-induced RNAi depletion of either Y14 (panel i), eIF4AIII (panel ii), or SF2/ASF (panel iii) in the PTCx strain does not rescue GFP expression in the affected embryos. As a control, depletion of Y14 (panel iv), eIF4AIII (panel v), and SF2/ASF (panel vi) does not prevent the GFP expression in the embryos of the wild-type strain. The RNAi-mediated depletion resulted in the expected phenotypes, as previously described (Longman et al. 2000, 2003). (B) Table summarizing the role of EJC components and splicing factors in NMD.

Figure 5.

An RNAi screen led to the identification of two novel NMD factors in C. elegans. (A, panel i) As a negative control, RNAi was induced with empty vector. (Panel ii) As a positive control, RNAi was induced with the smg-2 clone, which led to strong GFP expression within the PTCx strain. Depletion of the F20G4.1 clone (panel iii) and the Y37E11AM.1 clone (panel iv) resulted in increased GFP expression. (B, panels i,ii) The level of PTCx reporter mRNA was monitored by semiquantitative RT–PCR. In the PTCx strain, the level of reporter mRNA is very low (lane 2); however, it is significantly increased upon depletion of SMG-2. (Panels i,ii) Depletion of F20G4.1 or Y37E11AM.1 by RNAi led to an increase in PTCx mRNA level that is comparable with the depletion of SMG-2, a bona fide NMD factor (cf. lanes 4 and 6). A control mRNA encoding the splicing factor SRp20 is not altered by any of these treatments.

Figure 6.

Sequence conservation and functional domains of the newly characterized NMD factors. (A) Schematic representation of the human NAG, which is the homolog of C. elegans SMGL-1. Black boxes represent the regions of sequence conservation within proteins; the level of conservation is indicated. WD40 domains within the N terminus of the protein are indicated by brackets above the protein. (B) Schematic representation of the human DHX34, which is the homolog of C. elegans SMGL-2. Black boxes represent the regions of sequence conservation within proteins; the level of conservation is indicated. Functional domains associated with ATP-dependent RNA helicases (DEXDc, HELICc, PFAM:HA2, and PFAM:DUF 1605) are indicated by brackets above the protein.

Figure 7.

The human homologs of the novel C. elegans factors are required for NMD in human cells. (A) HeLa cells were mock-depleted, or depleted of UPF1, NAG, and DHX34 using two nonoverlapping siRNA pools. The level of knockdown of individual depletions was measured by real-time RT–PCR. The graph represents data of at least three independent experiments for each siRNA pool. For UPF1, the level of depletion was also assessed by Western blotting. (B) Depletion of both NAG and DHX34 genes, accomplished by two nonoverlapping siRNA pools, led to the increase of the β-globin reporter (β39) mRNA level. Depleted HeLa cells were transiently transfected with NMD β-globin reporter (β39), together with an EGFP vector as a transfection control. Total RNA was isolated 24 h post-transfection and the steady-state level of the reporter mRNA was measured by real-time RT–PCR. A schematic representation of the NMD β-globin reporter (β39) is below the graph.