Amyloids transmit acquired epigenetic feminizing information between generations.

In a recent article in Nature Cell Biology, Eroglu et al. link heritable, environment-induced epigenetic germline feminization to the accumulation, transmission, and replication of amyloid particles in Caenorhabditis elegans.

Reported transgenerational responses to in are not robust.

Here we report our attempt to replicate reports of transgenerational epigenetic inheritance in . Published results from multiple laboratories show that adults and their F1 embryos exposed to the pathogen show pathogen aversion behavior and a pathogen exposure-dependent increase in reporter gene expression. However, results from one group show persistence of the aversion behavior and elevated expression in the F2, F3, and F4 generations.

SID-4/NCK-1 is important for dsRNA import in Caenorhabditis elegans.

RNA interference is sequence-specific gene silencing triggered by double-stranded RNA. Systemic RNA interference is where double-stranded RNA, expressed or introduced into 1 cell, is transported to and initiates RNA interference in other cells. Systemic RNA interference is very efficient in Caenorhabditis elegans and genetic screens for systemic RNA interference-defective mutants have identified RNA transporters (SID-1, SID-2, and SID-5) and a signaling protein (SID-3).

Efficient Homologous Recombination in Mice Using Long Single Stranded DNA and CRISPR Cas9 Nickase.

The CRISPR/Cas9 nickase mutant is less prone to off-target double-strand (ds)DNA breaks than wild-type Cas9 because to produce dsDNA cleavage it requires two guide RNAs to target the nickase to nearby opposing strands. Like wild-type Cas9 lesions, these staggered lesions are repaired by either non-homologous end joining or, if a repair template is provided, by homologous recombination (HR). Here, we report very efficient (up to 100%) recovery of heterozygous insertions in produced by long (>300 nt), single-stranded DNA donor template-guided repair of paired-nickase lesions.

Intergenerational Transmission of Gene Regulatory Information in Caenorhabditis elegans.

Epigenetic mechanisms can stably maintain gene expression states even after the initiating conditions have changed. Often epigenetic information is transmitted only to daughter cells, but evidence is emerging, in both vertebrate and invertebrate systems, for transgenerational epigenetic inheritance (TEI), the transmission of epigenetic gene regulatory information across generations. Each new description of TEI helps uncover the properties, molecular mechanisms and biological roles for TEI.

SID-1 Domains Important for dsRNA Import in .

In the nematode , RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) spreads systemically to cause gene silencing throughout the organism and its progeny. We confirm that nematode SID-1 orthologs have dsRNA transport activity and demonstrate that the SID-1 paralog CHUP-1 does not transport dsRNA. Sequence comparison of these similar proteins, in conjunction with analysis of loss-of-function missense alleles, identifies several conserved 2-7 amino acid microdomains within the extracellular domain (ECD) that are important for dsRNA transport.

SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear.

SID-1 Functions in Multiple Roles To Support Parental RNAi in .

Systemic RNA interference (RNAi) in requires , , and Injected, expressed, or ingested double-stranded RNA (dsRNA) is transported between cells, enabling RNAi in most tissues, including the germline and progeny (parental RNAi). A recent report claims that parental RNAi also requires the yolk receptor Here, we characterize the role of the genes and in parental RNAi. We identify multiple independent paths for maternal dsRNA to reach embryos and initiate RNAi.

Stable Heritable Germline Silencing Directs Somatic Silencing at an Endogenous Locus.

The importance of transgenerationally inherited epigenetic states to organismal fitness remains unknown as well-documented examples are often not amenable to mechanistic analysis or rely on artificial reporter loci. Here we describe an induced silenced state at an endogenous locus that persists, at 100% transmission without selection, for up to 13 generations. This unusually persistent silencing enables a detailed molecular genetic analysis of an inherited epigenetic state.

Natural RNA interference directs a heritable response to the environment.

RNA interference can induce heritable gene silencing, but it remains unexplored whether similar mechanisms play a general role in responses to cues that occur in the wild. We show that transient, mild heat stress in the nematode Caenorhabditis elegans results in changes in messenger RNA levels that last for more than one generation. The affected transcripts are enriched for genes targeted by germline siRNAs downstream of the piRNA pathway, and worms defective for germline RNAi are defective for these heritable effects.

The RNA binding protein MEX-3 retains asymmetric activity in the early Caenorhabditis elegans embryo in the absence of asymmetric protein localization.

The RNA binding protein MEX-3 is required to restrict translation of pal-1, the Caenorhabditis elegans caudal homolog, to the posterior of the early embryo. MEX-3 is present uniformly throughout the newly fertilized embryo, but becomes depleted in the posterior by the 4-cell stage. This MEX-3 patterning requires the CCCH zinc-finger protein MEX-5, the RNA Recognition Motif protein SPN-4, and the kinase PAR-4.

Assays for direct and indirect effects of C. elegans endo-siRNAs.

Ever since the discovery of the first microRNAs in C. elegans, increasing numbers of endogenous small RNAs have been discovered. Endogenous siRNAs (endo-siRNAs) have emerged in the last few years as a largely independent class of small RNAs that regulate endogenous gene expression, with mechanisms distinct from those of piRNAs and miRNAs.

The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans.

RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C.

The nuclear argonaute NRDE-3 contributes to transitive RNAi in Caenorhabditis elegans.

The Caenorhabditis elegans nuclear RNA interference defective (Nrde) mutants were identified by their inability to silence polycistronic transcripts in enhanced RNAi (Eri) mutant backgrounds. Here, we report additional nrde-3-dependent RNAi phenomena that extend the mechanisms, roles, and functions of nuclear RNAi. We show that nrde-3 mutants are broadly RNAi deficient and that overexpressing NRDE-3 enhances RNAi.

SID-5 is an endosome-associated protein required for efficient systemic RNAi in C. elegans.

In the nematode C. elegans, RNAi silencing signals are efficiently taken up from the environment and transported between cells and tissues. Previous studies implicating endosomal proteins in systemic RNAi lack conclusive evidence.

Uptake of extracellular double-stranded RNA by SID-2.

Ingested dsRNAs trigger RNA interference (RNAi) in many invertebrates, including the nematode Caenorhabditis elegans. Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C.

A genomic bias for genotype-environment interactions in C. elegans.

The phenotype of an organism is determined by its genotype and environment. An interaction between these two arises from the differential effect of the environment on gene expression in distinct genotypes; however, the genomic properties identifying these are not well understood. Here we analyze the transcriptomes of five C.

Vampiric isolation of extracellular fluid from Caenorhabditis elegans.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems.

Two classes of silencing RNAs move between Caenorhabditis elegans tissues.

Organism-wide RNA interference (RNAi) is due to the transport of mobile silencing RNA throughout the organism, but the identities of these mobile RNA species in animals are unknown. Here, we present genetic evidence that both the initial double-stranded RNA (dsRNA), which triggers RNAi, and at least one dsRNA intermediate produced during RNAi can act as or generate mobile silencing RNA in C. elegans.

SID-1 is a dsRNA-selective dsRNA-gated channel.

Systemic RNAi in Caenorhabditis elegans requires the widely conserved transmembrane protein SID-1 to transport RNAi silencing signals between cells. When expressed in Drosophila S2 cells, C. elegans SID-1 enables passive dsRNA uptake from the culture medium, suggesting that SID-1 functions as a channel for the transport of double-stranded RNA (dsRNA).