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.[Keywords: Nonsense-mediated decay; smg genes; RNAi screens; exon junction complex; C. elegans] Supplemental material is available at http://www.genesdev.org. Nonsense-mediated mRNA decay (NMD) is a highly conserved surveillance mechanism present in all eukaryotes examined that prevents the synthesis of truncated proteins. It does so by promoting the degradation of mRNAs containing premature translation termination codons (PTCs) (for a recent review, see Maquat 2004). The functional importance of NMD is highlighted by the fact that this pathway targets for degradation a wide array of endogenous transcripts, as well as erroneously processed transcripts and transcripts carrying spontaneous mutations (for review, see Rehwinkel et al. 2006). It is proposed that NMD modulates the phenotypic outcome of many diseases, since ∼30% of inherited genetic disorders are caused by frameshift or nonsense mutations that generate premature termination codons (PTCs). Therefore, effectors of NMD are potential targets for therapeutic intervention (Frischmeyer and Dietz 1999;Holbrook et al. 2004).Genetic screens in the nematode Caenorhabditis elegans identified seven genes that are required for the degradation of nonsense mutant mRNAs of the unc-54 myosin heavy chain gene (Hodgkin et al. 1989;Pulak and Anderson 1993;Cali et al. 1999). In addition to their suppression-of-unc phenotype, these mutations cause abnormal morphogenesis of the male bursa and the hermaphrodite vulva, and accordingly these genes were termed smg-1-7 (for suppressor with morphological effect on genitalia). In the yeast Sac...

A genome-wide RNAi screen identifies genes regulating the formation of P bodies in C. elegans and their functions in NMD and RNAi

“…We found that all 83 RNAi clones identified above elevated the expression level of PTCx, in a magnitude similar to smgl-1 (Fig. 5H and 5I) (Longman et al, 2007). Thus, the identified 83 RNAi clones appear to suppress the degradation of PTC containing mRNAs in a gene-nonspecific manner.…”

“…We further examined the effect of the identified 83 RNAi clones on the expression of a previously characterized NMD target, PTCx (Longman et al, 2007). The PTCx reporter contains a GFP::LacZ fusion gene that is driven by a ubiquitous expressed promoter and a PTC is placed in the first exon of lacZ (lacZ containing four synthetic introns) (Longman et al, 2007).…”

“…The PTCx reporter contains a GFP::LacZ fusion gene that is driven by a ubiquitous expressed promoter and a PTC is placed in the first exon of lacZ (lacZ containing four synthetic introns) (Longman et al, 2007). Thus, gfp is only weakly expressed in animals carrying the PTCx transgene (Fig.…”

“…In mammalian cells, the exon junction complex (EJC), including Y14, Magoh, eIF4AIII and Barentsz, is required for recognizing PTC and triggering NMD (Isken and Maquat, 2007). In C. elegans and Drosophila, PTC recognition occurs independent of introns and consequently, component of EJC is dispensable for NMD (Mango, 2001;Longman et al, 2007). However, the core NMD machinery components, including UPF1, UPF2, UPF3, SMG1, SMG5 and SMG7, are evolutionarily conserved (Behm-Ansmant and Izaurralde, 2006;Isken and Maquat, 2007).…”

Studying gene function in Caenorhabditis elegans using RNA-mediated interference

“…Such surveys have identified proteins that regulate longevity/ageing [37][38][39][40][41][42], fat metabolism [43], transposon silencing [44], the DNA damage response [45], pronuclear migration during fertilization [46], germ cell apoptosis [47], co-suppression [48], the nonsensemediated mRNA decay pathway [49], RNAi [50] and those that protect against mutation [51]. Two other recent studies that address the regulation of cell migration and axon guidance are discussed here.…”