Search

RNAi pathways detect and silence foreign nucleic acids such as viruses as well as endogenous genes in many species. The phylogenetic profile across eukaryotes of proteins that mediate key steps in RNAi is correlated with the profiles of multiple mRNA splicing proteins and with intron number, suggesting that RNAi may surveil mRNA splicing to detect the divergent or absent introns of viruses. Here we examine the role of mRNA splicing in Caenorhabditis elegans RNAi. We found that viable null mutations in U1 and U2 small nuclear ribonucleic protein (snRNP)-specific splicing factor genes cause defects in RNAi. The U1A ortholog rnp-2 is required for normal ERGO-1 Argonaute class 26G siRNA biogenesis, trans-splicing of the eri-6/7 transcript, and targeting of poorly conserved gene transcripts by WAGO Argonaute class 22G siRNAs. We found that gene transcripts engaged by the siRNA-generating machinery are poorly conserved, possess few introns, and often have introns that are divergent from introns with strong consensus splicing sites found in highly conserved genes. We present biochemical evidence that RNAi targeted transcripts are tightly bound to spliceosomes. These findings suggest multiple layers of regulation by the spliceosome at early steps of small RNA-mediated gene silencing.

Hundreds of microRNAs (miRNAs) have been discovered in metazoans and plants, and understanding of their biogenesis has advanced dramatically; however, relatively little is known about the cofactors necessary for miRNA regulation of target gene expression. In Caenorhabditis elegans, the conserved miRNA let-7 and its paralogs, including mir-84, control the timing of stage-specific developmental events. To identify factors required for the activity of mir-84 and possibly other miRNAs, we screened for mutations that suppress the developmental defects caused by overexpression of mir-84. Mutations in the somi-1 gene prevent these defects without affecting the expression level of mir-84. Loss of somi-1 also causes phenotypes similar to deletion of mir-84, showing that somi-1 is necessary for the normal function of this miRNA. somi-1 encodes a zinc finger protein that localizes to nuclear foci and binds the promoters of let-60/RAS, lin-14, and lin-28, genes that may be targeted by mir-84 and similar miRNAs. Genetic evidence shows that somi-1 inhibits lin-14 and induction of the vulval precursors by the let-60/RAS pathway. Proteomic and genetic screens identified conserved chromatin-remodeling and homeodomain transcription factor complexes that work with somi-1 to regulate differentiation. Our results suggest that somi-1 coordinates a nuclear response that complements the activity of mir-84.

We report here the first genome-wide functional genomic screen for longevity genes. We systematically surveyed Caenorhabditis elegans genes using large-scale RNA interference (RNAi), and found that RNAi inactivation of 89 genes extend C. elegans lifespan. Components of the daf-2/insulin-like signaling pathway are recovered, as well as genes that regulate metabolism, signal transduction, protein turnover, and gene expression. Many of these candidate longevity genes are conserved across animal phylogeny. Genetic interaction analyses with the new longevity genes indicate that some act upstream of the daf-16/FOXO transcription factor or the sir2.1 protein deacetylase, and others function independently of daf-16/FOXO and sir2.1, and might define new pathways to regulate lifespan.

The activity of the DAF-2 insulin-like receptor is required forCaenorhabditis elegans reproductive growth and normal adult life span. Informatic analysis identified 37 C. elegans genes predicted to encode insulin-like peptides. Many of these genes are divergent insulin superfamily members, and many are clustered, indicating recent diversification of the family. The ins genes are primarily expressed in neurons, including sensory neurons, a subset of which are required for reproductive development. Structural predictions and likely C-peptide cleavage sites typical of mammalian insulins suggest that ins-1 is most closely related to insulin. Overexpression of ins-1, or expression of human insulin under the control of ins-1 regulatory sequences, causes partially penetrant arrest at the dauer stage and enhances dauer arrest in weakdaf-2 mutants, suggesting that INS-1 and human insulin antagonize DAF-2 insulin-like signaling. A deletion of theins-1 coding region does not enhance or suppress dauer arrest, indicating a functional redundancy among the 37 ins genes. Of five other ins genes tested, the only other one bearing a predicted C peptide also antagonizes daf-2 signaling, whereas four ins genes without a C peptide do not, indicating functional diversity within the ins family.

The Caenorhabditis elegans AWA, AWB, and AWC olfactory neurons are each required for the recognition of a specific subset of volatile odorants. lim-4 mutants express an AWC reporter gene inappropriately in the AWB olfactory neurons and fail to express an AWB reporter gene. The AWB cells are morphologically transformed toward an AWC fate in lim-4 mutants, adopting cilia and axon morphologies characteristic of AWC. AWB function is also transformed in these mutants: Rather than mediating the repulsive behavioral responses appropriate for AWB, the AWB neurons mediate attractive responses, like AWC. LIM-4 is a predicted LIM homeobox gene that is expressed in AWB and a few other head neurons. Ectopic expression of LIM-4 in the AWC neuron pair is sufficient to force those cells to adopt an AWB fate. The AWA nuclear hormone receptor ODR-7 described previously also represses AWC genes, as well as inducing AWA genes. We propose that the LIM-4 and ODR-7 transcription factors function to diversify C. elegans olfactory neuron identities, driving them from an AWC-like state into alternative fates.

In response to environmental signals, Caenorhabditis elegans has two distinct life-cycles—reproductive, in which the animals rapidly grow to the adult reproductive stage, and dauer, in which the animals arrest development at the anatomically and metabolically distinctive third-larval dauer stage (Riddle and Albert 1997). Environmental conditions are sensed in chemosensory neurons (Bargmann and Horvitz 1991), which couple to a transforming growth factor β (TGF-β) signaling pathway (Georgi et al. 1990; Estevez et al. 1993; Ren et al. 1996) as well as an insulin-related signaling pathway (Morris et al. 1996; Kimura et al. 1997) to trigger changes in the development of the many tissues remodeled in dauer larvae (Riddle and Albert 1997). Mutations in daf-7 [a TGF-β homolog (Ren et al. 1996)], daf-4 [a type II TGF-β receptor (Estevez et al. 1993)], daf-1 [a type I TGF-β receptor (Georgi et al. 1990)], daf-8, and daf-14 [Smad (for small and mad) homologs (A. Estevez and D.L. Riddle, pers. comm.; T. Inoue and J. Thomas, pers. comm.)] cause constitutive arrest at the dauer stage even in conditions that promote reproductive growth. These genes constitute a neuronal signaling pathway that is active during reproductive development. The DAF-7 TGF-β signal is produced by the sensory neuron ASI during reproductive development, whereas daf-7 expression in this neuron is inhibited during dauer-inducing conditions (Ren et al. 1996; Schackwitz et al. 1996). DAF-7 binding to the DAF-1/DAF-4 receptors has been suggested to activate the DAF-8/DAF-14 Smads to promote a commitment to reproductive growth (Ren et al. 1996; A. Estevez and D.L. Riddle, pers. comm.; T. Inoue and J. Thomas, pers. comm.). A model for signaling by TGF-β and related receptors has emerged recently. Binding of ligand induces dimerization of receptors to produce an active signaling kinase (Attisano et al. 1994; Miyazano et al. 1994). This complex phosphorylates cytoplasmic Smad proteins and causes them to relocate to the nucleus, where they act as transcription factors (Chen et al. 1996; Liu et al. 1996; Mascias-Silva et al. 1996; Chen et al. 1997; Kim et al. 1997; Kretzschmar et al. 1997). The Smad protein phosphorylated is determined by the identity of the complex, for example, BMP receptors phosphorylate Smad1 (Kretzschmar et al. 1997), whereas TGF-β and activin receptors target Smad2 or Smad3 (Chen et al. 1996; Chen et al. 1996; Lagna et al. 1996; Mascias-Silva et al. 1996; Zhang et al. 1996, 1997). All of the above Smads interact with DPC4, a member of a second class of Smad proteins, and all three pathways are potentiated by DPC4 (Lagna et al. 1996; Mascias-Silva et al. 1996; Zhang et al. 1996, 1997; Kretzschmar et al. 1997); therefore, DPC4 is thought to be a general cofactor for pathway-specific Smads such as Smad1, Smad2, and Smad3. The small TGF-β-related pathway in C. elegans also requires both a DPC4-like Smad (sma-4) and two other Smads more closely related to Smad-1 (sma-2 and sma-3) (Savage et al. 1996). Drosophila melanogaster also has Mad, which resembles Smad1, and a DPC4 homolog as well (P. Das and R. Padgett, pers. comm.). The requirement for these two basic types of Smads may be conserved in TGF-β related-pathways across metazoan phylogeny. Because Smad proteins are positive transducers of signals, the biological effect of overexpressing Smads is the same as overexpressing or activating receptors (Baker and Harland 1996; Graff et al. 1996), and the effect of Smad mutations mimics the effect of loss-of-function mutations in the receptors (Raftery et al. 1995; Sekelsky et al. 1995; Savage et al. 1996). And, although screens for enhancers of TGF-β pathway mutations have been successful (Raftery et al. 1995), suppressors of TGF-β pathway mutants have not been identified, except in the C. elegans dauer pathway (Riddle et al. 1981; Thomas et al. 1993). daf-3 is a unique TGF-β pathway gene in that it is antagonized, rather than activated by the daf-7 TGF-β-related pathway. The dauer-constitutive phenotypes of mutations in the DAF-7 signal transduction pathway genes (including putative null mutations, see below) are suppressed fully by mutations in daf-3 (Riddle et al. 1981; Vowels and Thomas 1992). These genetic data indicate that in the absence of DAF-7 signaling, the lack of antagonism releases DAF-3 activity to induce dauer arrest. In this report, we show that daf-3 encodes a novel Smad, which is antagonized rather than activated by a TGF-β-related pathway. We show that DAF-3 and the TGF-β receptor DAF-4 are expressed widely in tissues that remodel during dauer development, and therefore, may transduce DAF-7 neuroendocrine signals throughout the body to regulate tissue metabolism and development. We show that a DAF-3/GFP fusion protein is nuclear localized and perturbs dauer arrest when mutations are directed to a conserved domain in the carboxy-terminal region, and that DAF-3/GFP fusion proteins are associated with mitotic chromosomes, indicating that DAF-3 functions in the nucleus. The DAF-3 expression level and subcellular localization is independent of upstream DAF-7 signal transduction activity, indicating that the regulation of DAF-3 by DAF-7 signaling may occur downstream of nuclear localization. We propose a model for regulation of DAF-3 activity by the DAF-8/DAF-14 Smads.

The heterchronic gene lin-14 controls the temporal sequence of developmental events in the Caenorhabditis elegans postembryonic cell lineage. It encodes a nuclear protein that normally is present in most somatic cells of late embryos and L1 larvae but is absent at later stages. Two lin-14 gain-of-function mutations delete 3'-untranslated sequences causing an inappropriately high level of the lin-14 nuclear protein late in development. These mutations identify a negative regulatory element that controls the formation of the lin-14 protein temporal gradient. The 21-kb lin-14 gene is differentially spliced to generate three lin-14 transcripts that encode protein products with variable amino-terminal regions and a constant carboxy-terminal region. The sequence of the gene revealed no protein sequence similarity to any proteins in various data bases.