RNA interference (RNAi) is a broadly used reverse genetics method in C. elegans. Unfortunately, RNAi does not inhibit all genes. We show that loss of function of a putative RNA-directed RNA polymerase (RdRP) of C. elegans, RRF-3, results in a substantial enhancement of sensitivity to RNAi in diverse tissues. This is particularly striking in the nervous system; neurons that are generally refractory to RNAi in a wild-type genetic background can respond effectively to interference in an rrf-3 mutant background. These data provide the first indication of physiological negative modulation of the RNAi response and implicate an RdRP-related factor in this effect. The rrf-3 strain can be useful to study genes that, in wild-type, do not show a phenotype after RNAi, and it is probably the strain of choice for genome-wide RNAi screens.

Recent genetical genomics studies have provided intimate views on gene regulatory networks. Gene expression variations between genetically different individuals have been mapped to the causal regulatory regions, termed expression quantitative trait loci. Whether the environment-induced plastic response of gene expression also shows heritable difference has not yet been studied. Here we show that differential expression induced by temperatures of 16 °C and 24 °C has a strong genetic component in Caenorhabditis elegans recombinant inbred strains derived from a cross between strains CB4856 (Hawaii) and N2 (Bristol). No less than 59% of 308 trans-acting genes showed a significant eQTL-by-environment interaction, here termed plasticity quantitative trait loci. In contrast, only 8% of an estimated 188 cis-acting genes showed such interaction. This indicates that heritable differences in plastic responses of gene expression are largely regulated in trans. This regulation is spread over many different regulators. However, for one group of trans-genes we found prominent evidence for a common master regulator: a transband of 66 coregulated genes appeared at 24 °C. Our results suggest widespread genetic variation of differential expression responses to environmental impacts and demonstrate the potential of genetical genomics for mapping the molecular determinants of phenotypic plasticity.

Histone H3 lysine 9 (H3K9) methylation is a conserved modification that generally represses transcription. In Caenorhabditis elegans it is enriched on silent tissue-specific genes and repetitive elements. In met-2 set-25 double mutants, which lack all H3K9 methylation (H3K9me), embryos differentiate normally, although mutant adults are sterile owing to extensive DNA-damage-driven apoptosis in the germ line. Transposons and simple repeats are derepressed in both germline and somatic tissues. This unprogrammed transcription correlates with increased rates of repeat-specific insertions and deletions, copy number variation, R loops and enhanced sensitivity to replication stress. We propose that H3K9me2 or H3K9me3 stabilizes and protects repeat-rich genomes by suppressing transcription-induced replication stress.

Small RNA molecules participate in a variety of activities in the cell: in a process known as RNA interference (RNAi), double-stranded RNA triggers the degradation of messenger RNA that has a matching sequence; the small RNA intermediates of this process can also modify gene expression in the nucleus. Here we show that a single episode of RNAi in the nematode Caenorhabditis elegans can induce transcriptional silencing effects that are inherited indefinitely in the absence of the original trigger. Our findings may prove useful in the ongoing development of RNAi to treat disease.

Genomes contain many sequences that are intrinsically difficult to replicate. Tracts of tandem guanines, for instance, have the potential to adopt stable G-quadruplex structures, which are prone to cause genome alterations. Here we describe G4 DNA-induced mutagenesis in Caenorhabditis elegans and identify a non-canonical DNA break repair mechanism that generates deletions characterized by an extremely narrow size distribution, minimal homology of exactly one nucleotide at the junctions, and by the occasional presence of templated insertions. This typical mutation profile is fully dependent on the A-family polymerase Theta, the absence of which leads to profound loss of sequences surrounding G4 motifs. Theta-mediated end-joining prevails over non-homologous end joining and homologous recombination and prevents genomic havoc at replication fork barriers at the expense of small deletions. G4 DNA-induced deletions also manifest in the genomes of wild isolates of C. elegans, indicating a protective role for this pathway during evolution.