Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference

Background

Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms.

Results

We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain.

Conclusion

A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.     

RDE-2 interacts with MUT-7 to mediate RNA interference in Caenorhabditis elegans

In Caenorhabditis elegans, the activity of transposable elements is repressed in the germline. One of the mechanisms involved in this repression is RNA interference (RNAi), a process in which dsRNA targets cleavage of mRNAs in a sequence-specific manner. The first gene found to be involved in RNAi and transposon silencing in C.elegans is mut-7, a gene encoding a putative exoribonuclease. Here, we show that the MUT-7 protein resides in complexes of ~250 kDa in the nucleus and in the cytosol. In addition, we find that upon triggering of RNAi the cytosolic MUT-7 complex increases in size. This increase is independent of the presence of target RNA, but does depend on the presence of RDE-1 and RDE-4, two proteins involved in small interfering RNA (siRNA) production. Finally, using a yeast two-hybrid screen, we identified RDE-2/MUT-8 as one of the other components of this complex. This protein is encoded by the rde-2/mut-8 locus, previously implicated in RNAi and transposon silencing. Using genetic complementation analysis, we show that the interaction between these two proteins is required for efficient RNAi in vivo. Together these data support a role for the MUT-7/RDE-2 complex downstream of siRNA formation, but upstream of siRNA mediated target RNA recognition, possibly indicating a role in the siRNA amplification step.     

PAB-1, a Caenorhabditis elegans Poly(A)-Binding Protein,Regulates mRNA Metabolism in germline by Interacting with CGH-1 and CAR-1

Poly(A)-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P)-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.     

Antiviral RNA Silencing Initiated in the Absence of RDE-4, a Double-Stranded RNA Binding Protein,in Caenorhabditis elegans

Small interfering RNAs (siRNAs) processed from double-stranded RNA (dsRNA) of virus origins mediate potent antiviral defense through a process referred to as RNA interference (RNAi) or RNA silencing in diverse organisms. In the simple invertebrate Caenorhabditis elegans, the RNAi process is initiated by a single Dicer, which partners with the dsRNA binding protein RDE-4 to process dsRNA into viral siRNAs (viRNAs). Notably, in C. elegans this RNA-directed viral immunity (RDVI) also requires a number of worm-specific genes for its full antiviral potential. One such gene is rsd-2 (RNAi spreading defective 2), which was implicated in RDVI in our previous studies. In the current study, we first established an antiviral role by showing that rsd-2 null mutants permitted higher levels of viral RNA accumulation, and that this enhanced viral susceptibility was reversed by ectopic expression of RSD-2. We then examined the relationship of rsd-2 with other known components of RNAi pathways and established that rsd-2 functions in a novel pathway that is independent of rde-4 but likely requires the RNA-dependent RNA polymerase RRF-1, suggesting a critical role for RSD-2 in secondary viRNA biogenesis, likely through coordinated action with RRF-1. Together, these results suggest that RDVI in the single-Dicer organism C. elegans depends on the collective actions of both RDE-4-dependent and RDE-4-independent mechanisms to produce RNAi-inducing viRNAs. Our study reveals, for the first time, a novel siRNA-producing mechanism in C. elegans that bypasses the need for a dsRNA-binding protein.     

Comparative functional characterization of the CSR-1 22G-RNA pathway in Caenorhabditis nematodes

As a champion of small RNA research for two decades, Caenorhabditis elegans has revealed the essential Argonaute CSR-1 to play key nuclear roles in modulating chromatin, chromosome segregation and germline gene expression via 22G-small RNAs. Despite CSR-1 being preserved among diverse nematodes, the conservation and divergence in function of the targets of small RNA pathways remains poorly resolved. Here we apply comparative functional genomic analysis between C. elegans and Caenorhabditis briggsae to characterize the CSR-1 pathway, its targets and their evolution. C. briggsae CSR-1-associated small RNAs that we identified by immunoprecipitation-small RNA sequencing overlap with 22G-RNAs depleted in cbr-csr-1 RNAi-treated worms. By comparing 22G-RNAs and target genes between species, we defined a set of CSR-1 target genes with conserved germline expression, enrichment in operons and more slowly evolving coding sequences than other genes, along with a small group of evolutionarily labile targets. We demonstrate that the association of CSR-1 with chromatin is preserved, and show that depletion of cbr-csr-1 leads to chromosome segregation defects and embryonic lethality. This first comparative characterization of a small RNA pathway in Caenorhabditis establishes a conserved nuclear role for CSR-1 and highlights its key role in germline gene regulation across multiple animal species.     

An RIG-I-Like RNA Helicase Mediates Antiviral RNAi Downstream of Viral siRNA Biogenesis in Caenorhabditis elegans

Dicer ribonucleases of plants and invertebrate animals including Caenorhabditis elegans recognize and process a viral RNA trigger into virus-derived small interfering RNAs (siRNAs) to guide specific viral immunity by Argonaute-dependent RNA interference (RNAi). C. elegans also encodes three Dicer-related helicase (drh) genes closely related to the RIG-I-like RNA helicase receptors which initiate broad-spectrum innate immunity against RNA viruses in mammals. Here we developed a transgenic C. elegans strain that expressed intense green fluorescence from a chromosomally integrated flock house virus replicon only after knockdown or knockout of a gene required for antiviral RNAi. Use of the reporter nematode strain in a feeding RNAi screen identified drh-1 as an essential component of the antiviral RNAi pathway. However, RNAi induced by either exogenous dsRNA or the viral replicon was enhanced in drh-2 mutant nematodes, whereas exogenous RNAi was essentially unaltered in drh-1 mutant nematodes, indicating that exogenous and antiviral RNAi pathways are genetically distinct. Genetic epistatic analysis shows that drh-1 acts downstream of virus sensing and viral siRNA biogenesis to mediate specific antiviral RNAi. Notably, we found that two members of the substantially expanded subfamily of Argonautes specific to C. elegans control parallel antiviral RNAi pathways. These findings demonstrate both conserved and unique strategies of C. elegans in antiviral defense.     

The C. elegans TPR Containing Protein,TRD-1, Regulates Cell Fate Choice in the Developing Germ Line and Epidermis

Correct cell fate choice is crucial in development. In post-embryonic development of the hermaphroditic Caenorhabitis elegans, distinct cell fates must be adopted in two diverse tissues. In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes. In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam) or differentiated (hypodermal or neuronal) fate. We have isolated a novel conserved C. elegans tetratricopeptide repeat containing protein, TRD-1, which is essential for cell fate determination in both the germline and the developing epidermis and has homologs in other species, including humans (TTC27). We show that trd-1(RNAi) and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate. In the germline, trd-1 RNAi results in a strong masculinization phenotype, as well as defects in the mitosis to meiosis switch. Our data suggests that trd-1 acts downstream of tra-2 but upstream of fem-3 in the germline sex determination pathway, and exhibits a constellation of phenotypes in common with other Mog (masculinization of germline) mutants. Thus, trd-1 is a new player in both the somatic and germline cell fate determination machinery, suggestive of a novel molecular connection between the development of these two diverse tissues.     

Evolution of dnmt-2 and mbd-2-like genes in the free-living nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae

Whole genome sequencing of several metazoan model organisms provides a platform for studying genome evolution. How representative are the genomes of these model organisms for their respective phyla? Within nematodes, for example, the free-living soil nematode Caenorhabditis elegans is a highly derived species with unusual genomic characters, such as a reduced Hox cluster (Curr. Biol., 13, 37–40) and the absence of a Hedgehog signaling system. Here, we describe the recent loss of a DNA methyltransferase-2 gene (dnmt-2) in C.elegans. A dnmt-2-like gene is present in the satellite model organism Pristionchus pacificus, another free-living nematode that diverged from C.elegans 200–300 million years ago. In contrast, C.elegans, Caenorhabditis briggsae and P.pacificus all contain an mbd-2-like gene, which encodes another essential component of the methylation system of higher animals and fungi. Cel-mbd-2 is expressed throughout development and RNA interference (RNAi) experiments result in variable phenotypes. In contrast, Cbr-mbd-2 RNAi results in paralyzed larval or adult worms suggesting recent changes of gene function within the genus Caenorhabditis. We speculate that both genes were part of an ancestral DNA methylation system in nematodes and that gene loss and sequence divergence have abolished DNA methylation in C.elegans.