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 growing body of evidence supports the coordination of pre-mRNA processing and transcriptional regulation. We demonstrate here that mammalian PRP4 kinase (PRP4K) is associated with complexes involved in both of these processes. PRP4K is implicated in pre-mRNA splicing as the homologue of the Schizosaccharomyces pombe pre-mRNA splicing kinase Prp4p, and it is enriched in SC35-containing nuclear splicing speckles. RNA interference of Caenorhabditis elegans PRP4K indicates that it is essential in metazoans. In support of a role for PRP4K in pre-mRNA splicing, we identified PRP6, SWAP, and pinin as interacting proteins and demonstrated that PRP4K is a U5 snRNP-associated kinase. In addition, BRG1 and N-CoR, components of nuclear hormone coactivator and corepressor complexes, also interact with PRP4K. PRP4K coimmunoprecipitates with N-CoR, BRG1, pinin, and PRP6, and we present data suggesting that PRP6 and BRG1 are substrates of this kinase. Lastly, PRP4K, BRG1, and PRP6 can be purified as components of the N-CoR-2 complex, and affinity-purified PRP4K/N-CoR complexes exhibit deacetylase activity. We suggest that PRP4K is an essential kinase that, in association with the both U5 snRNP and N-CoR deacetylase complexes, demonstrates a possible coordination of pre-mRNA splicing with chromatin remodeling events involved in transcriptional regulation.The regulation of eukaryotic gene expression involves the modulation of chromatin structure and the coordinated transcription and splicing of mRNA. Both histone modification (61) and chromatin remodeling modulate the access of the transcriptional apparatus to chromatin (14, 44). The SWI/SNF proteins are the archetypal family of ATPases involved in remodeling chromatin, and members of this family in both yeast (Snf2) and mammals (BRG1) have been isolated in association with the RNA polymerase II holoenzyme complex (45, 59). Once transcription has been initiated it is thought that premRNA splicing occurs cotranscriptionally (4,22). A number of recent studies have also shown links between corepressor and coactivator complexes and pre-mRNA splicing (e.g., PSF [40] and PGC1 [42], respectively), which suggests yet another level of coordination between the regulation of gene expression and pre-mRNA splicing.If chromatin remodeling and pre-mRNA splicing occur in coordination with transcription, there may be proteins that either play a direct role in both chromatin structure and splicing and/or interact with factors involved in each process. Previously, we isolated a murine gene-trap protein, designated CT143 (56), which is similar to Schizosaccharomyces pombe Prp4 kinase (17). Prp4p is essential for pre-mRNA splicing, as demonstrated by the accumulation of unspliced pre-mRNA at the restrictive temperature in yeast carrying a temperaturesensitive (ts) mutation in prp4 (2). Prp4p interacts genetically with other S. pombe splicing proteins, including the non-SR splicing proteins Prp1p (a homologue of Saccharomyces cerevisiae Prp6p) and Prp5p (51). Multiple rounds of phosphorylation and d...

The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR‐related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons but also regulates the abundance of cellular RNAs. We sought to identify transcripts that are regulated by two novel NMD factors, DHX34 and neuroblastoma amplified sequence (NBAS), which were identified in a genome-wide RNA interference screen in Caenorhabditis elegans and later shown to mediate NMD in vertebrates. We performed microarray expression profile analysis in human cells, zebrafish embryos and C. elegans that were individually depleted of these factors. Our analysis revealed that a significant proportion of genes are co-regulated by DHX34, NBAS and core NMD factors in these three organisms. Further analysis indicates that NMD modulates cellular stress response pathways and membrane trafficking across species. Interestingly, transcripts encoding different NMD factors were sensitive to DHX34 and NBAS depletion, suggesting that these factors participate in a conserved NMD negative feedback regulatory loop, as was recently described for core NMD factors. In summary, we find that DHX34 and NBAS act in concert with core NMD factors to co-regulate a large number of endogenous RNA targets. Furthermore, the conservation of a mechanism to tightly control NMD homeostasis across different species highlights the importance of the NMD response in the control of gene expression.