Next-Gen Learning: The C. elegans Approach.

In this issue, Moore et al. and Posner et al., provide evidence for how the activity of the nervous system in C. elegans results in gene expression changes in the germline to pass on parental experiences and learned behavior to their progeny.

Mechanisms of epigenetic regulation by C. elegans nuclear RNA interference pathways.

RNA interference (RNAi) is a highly conserved gene regulatory phenomenon whereby Argonaute/small RNA (AGO/sRNA) complexes target transcripts by antisense complementarity to modulate gene expression. While initially appreciated as a cytoplasmic process, RNAi can also occur in the nucleus where AGO/sRNA complexes are recruited to nascent transcripts. Nuclear AGO/sRNA complexes recruit co-factors that regulate transcription by inhibiting RNA Polymerase II, modifying histones, compacting chromatin and, in some organisms, methylating DNA. C. elegans has a longstanding history in unveiling the mechanisms of RNAi and has become an outstanding model to delineate the mechanisms underlying nuclear RNAi. In this review we highlight recent discoveries in the field of nuclear RNAi in C. elegans and the roles of nuclear RNAi in the regulation of gene expression, chromatin organization, genome stability, and transgenerational epigenetic inheritance.

CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs.

We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.

The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.

RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1-interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci but does not downregulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans.

Temperature-dependent Small RNA Expression Depends on Wild Genetic Backgrounds of Caenorhabditis briggsae.

Geographically distinct populations can adapt to the temperature conditions of their local environment, leading to temperature-dependent fitness differences between populations. Consistent with local adaptation, phylogeographically distinct Caenorhabditis briggsae nematodes show distinct fitness responses to temperature. The genetic mechanisms underlying local adaptation, however, remain unresolved. To investigate the potential role of small noncoding RNAs in genotype-specific responses to temperature, we quantified small RNA expression using high-throughput sequencing of C. briggsae nematodes from tropical and temperate strain genotypes reared under three temperature conditions (14 °C, 20 °C, and 30 C). Strains representing both tropical and temperate regions showed significantly lower expression of PIWI-interacting RNAs (piRNAs) at high temperatures, primarily mapping to a large ∼7 Mb long piRNA cluster on chromosome IV. We also documented decreased expression of 22G-RNAs antisense to protein-coding genes and other genomic features at high rearing temperatures for the thermally-intolerant temperate strain genotype, but not for the tropical strain genotype. Reduced 22G-RNA expression was widespread along chromosomes and among feature types, indicative of a genome-wide response. Targets of the EGO-1/CSR-1 22G-RNA pathway were most strongly impacted compared with other 22G-RNA pathways, implicating the CSR-1 Argonaute and its RNA-dependent RNA polymerase EGO-1 in the genotype-dependent modulation of C. briggsae 22G-RNAs under chronic thermal stress. Our work suggests that gene regulation via small RNAs may be an important contributor to the evolution of local adaptations.

Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility.

The Caenorhabditis elegans genome encodes nineteen functional Argonaute proteins that use 22G-RNAs, 26G-RNAs, miRNAs or piRNAs to regulate target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG into the long (CSR-1a) and short (CSR-1b) isoforms, we found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR-1a plays tissue specific functions. During spermatogenesis, CSR-1a integrates into an sRNA regulatory network including ALG-3, ALG-4 and WAGO-10 that is necessary for fertility at 25°C. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. Our findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.

Secretion of an Argonaute protein by a parasitic nematode and the evolution of its siRNA guides.

Extracellular RNA has been proposed to mediate communication between cells and organisms however relatively little is understood regarding how specific sequences are selected for export. Here, we describe a specific Argonaute protein (exWAGO) that is secreted in extracellular vesicles (EVs) released by the gastrointestinal nematode Heligmosomoides bakeri, at multiple copies per EV. Phylogenetic and gene expression analyses demonstrate exWAGO orthologues are highly conserved and abundantly expressed in related parasites but highly diverged in free-living genus Caenorhabditis. We show that the most abundant small RNAs released from the nematode parasite are not microRNAs as previously thought, but rather secondary small interfering RNAs (siRNAs) that are produced by RNA-dependent RNA Polymerases. The siRNAs that are released in EVs have distinct evolutionary properties compared to those resident in free-living or parasitic nematodes. Immunoprecipitation of exWAGO demonstrates that it specifically associates with siRNAs from transposons and newly evolved repetitive elements that are packaged in EVs and released into the host environment. Together this work demonstrates molecular and evolutionary selectivity in the small RNA sequences that are released in EVs into the host environment and identifies a novel Argonaute protein as the mediator of this.

Genome structure predicts modular transcriptome responses to genetic and environmental conditions.

Understanding the plasticity, robustness and modularity of transcriptome expression to genetic and environmental conditions is crucial to deciphering how organisms adapt in nature. To test how genome architecture influences transcriptome profiles, we quantified expression responses for distinct temperature-adapted genotypes of the nematode Caenorhabditis briggsae when exposed to chronic temperature stresses throughout development. We found that 56% of the 8,795 differentially expressed genes show genotype-specific changes in expression in response to temperature (genotype-by-environment interactions, GxE). Most genotype-specific responses occur under heat stress, indicating that cold vs. heat stress responses involve distinct genomic architectures. The 22 co-expression modules that we identified differ in their enrichment of genes with genetic vs. environmental vs. interaction effects, as well as their genomic spatial distributions, functional attributes and rates of molecular evolution at the sequence level. Genes in modules enriched for simple effects of either genotype or temperature alone tend to evolve especially rapidly, consistent with disproportionate influence of adaptation or weaker constraint on these subsets of loci. Chromosome-scale heterogeneity in nucleotide polymorphism, however, rather than the scale of individual genes predominates as the source of genetic differences among expression profiles, and natural selection regimes are largely decoupled between coding sequences and noncoding flanking sequences that contain cis-regulatory elements. These results illustrate how the form of transcriptome modularity and genome structure contribute to predictable profiles of evolutionary change.

Todos Santos small RNA symposium.

Worm biologists from the United States, Canada, and the United Kingdom gathered at the Colorado State University Todos Santos Center in Baja California Sur, Mexico, April 3-5, 2019 for the Todos Santos Small RNA Symposium. Meeting participants, many of whom were still recovering from the bomb cyclone that struck a large swath of North America just days earlier, were greeted by the warmth and sunshine that is nearly ubiquitous in the sleepy seaside town of Todos Santos. With only 24 speakers, the meeting had the sort of laid-back vibe you might expect amongst the palm trees and ocean breeze of the Pacific coast of Mexico. The meeting started with tracing the laboratory lineages of participants. Not surprisingly, the most common parental lineages represented at the meeting were Dr. Craig Mello, Dr. Gary Ruvkun, and Dr. Victor Ambros, whom, together with Dr. Andy Fire and Dr. David Baulcombe, pioneered the small RNA field. In sad irony, on the closing day of the meeting, participants were met with the news of Dr. Sydney Brenner's passing. By establishing the worm, Caenorhabditis elegans, as a model system Dr. Brenner paved the way for much of the research discussed here.

Comparative genomic analysis of upstream miRNA regulatory motifs in Caenorhabditis.

MicroRNAs (miRNAs) comprise a class of short noncoding RNA molecules that play diverse developmental and physiological roles by controlling mRNA abundance and protein output of the vast majority of transcripts. Despite the importance of miRNAs in regulating gene function, we still lack a complete understanding of how miRNAs themselves are transcriptionally regulated. To fill this gap, we predicted regulatory sequences by searching for abundant short motifs located upstream of miRNAs in eight species of Caenorhabditis nematodes. We identified three conserved motifs across the Caenorhabditis phylogeny that show clear signatures of purifying selection from comparative genomics, patterns of nucleotide changes in motifs of orthologous miRNAs, and correlation between motif incidence and miRNA expression. We then validated our predictions with transgenic green fluorescent protein reporters and site-directed mutagenesis for a subset of motifs located in an enhancer region upstream of let-7 We demonstrate that a CT-dinucleotide motif is sufficient for proper expression of GFP in the seam cells of adult C. elegans, and that two other motifs play incremental roles in combination with the CT-rich motif. Thus, functional tests of sequence motifs identified through analysis of molecular evolutionary signatures provide a powerful path for efficiently characterizing the transcriptional regulation of miRNA genes.

Examining the intersection between splicing, nuclear export and small RNA pathways.

BACKGROUND: Nuclear Argonaute/small RNA pathways in a variety of eukaryotic species are generally known to regulate gene expression via chromatin modulation and transcription attenuation in a process known as transcriptional gene silencing (TGS). However, recent data, including genetic screens, phylogenetic profiling, and molecular mechanistic studies, also point to a novel and emerging intersection between the splicing and nuclear export machinery with nuclear Argonaute/small RNA pathways in many organisms. SCOPE OF REVIEW: In this review, we summarize the field's current understanding regarding the relationship between splicing, export and small RNA pathways, and consider the biological implications for coordinated regulation of transcripts by these pathways. We also address the importance and available approaches for understanding the RNA regulatory logic generated by the intersection of these particular pathways in the context of synthetic biology. MAJOR CONCLUSIONS: The interactions between various eukaryotic RNA regulatory pathways, particularly splicing, nuclear export and small RNA pathways provide a type of combinatorial code that informs the identity ("self" versus "non-self") and dictates the fate of each transcript in a cell. Although the molecular mechanisms for how splicing and nuclear export impact small RNA pathways are not entirely clear at this early stage, the links between these pathways are widespread across eukaryotic phyla. GENERAL SIGNIFICANCE: The link between splicing, nuclear export, and small RNA pathways is emerging and establishes a new frontier for understanding the combinatorial logic of gene regulation across species that could someday be harnessed for therapeutic, biotechnology and agricultural applications. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.

Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the Dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that, in the germline, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage structures called P granules. WAGO-1 silences certain genes, transposons, pseudogenes, and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest additional regulatory functions for small RNAs.

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.

PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans.

In metazoans, Piwi-related Argonaute proteins have been linked to germline maintenance, and to a class of germline-enriched small RNAs termed piRNAs. Here we show that an abundant class of 21 nucleotide small RNAs (21U-RNAs) are expressed in the C. elegans germline, interact with the C. elegans Piwi family member PRG-1, and depend on PRG-1 activity for their accumulation. The PRG-1 protein is expressed throughout development and localizes to nuage-like structures called P granules. Although 21U-RNA loci share a conserved upstream sequence motif, the mature 21U-RNAs are not conserved and, with few exceptions, fail to exhibit complementarity or evidence for direct regulation of other expressed sequences. Our findings demonstrate that 21U-RNAs are the piRNAs of C. elegans and link this class of small RNAs and their associated Piwi Argonaute to the maintenance of temperature-dependent fertility.

Epstein-Barr virus EBNA1 protein regulates viral latency through effects on let-7 microRNA and dicer.

UNLABELLED: The EBNA1 protein of Epstein-Barr virus (EBV) plays multiple roles in EBV latent infection, including altering cellular pathways relevant for cancer. Here we used microRNA (miRNA) cloning coupled with high-throughput sequencing to identify the effects of EBNA1 on cellular miRNAs in two nasopharyngeal carcinoma cell lines. EBNA1 affected a small percentage of cellular miRNAs in both cell lines, in particular, upregulating multiple let-7 family miRNAs, including let-7a. The effects of EBNA1 on let-7a were verified by demonstrating that EBNA1 silencing in multiple EBV-positive carcinomas downregulated let-7a. Accordingly, the let-7a target, Dicer, was found to be partially downregulated by EBNA1 expression (at the mRNA and protein levels) and upregulated by EBNA1 silencing in EBV-positive cells. Reporter assays based on the Dicer 3' untranslated region with and without let-7a target sites indicated that the effects of EBNA1 on Dicer were mediated by let-7a. EBNA1 was also found to induce the expression of let-7a primary RNAs in a manner dependent on the EBNA1 transcriptional activation region, suggesting that EBNA1 induces let-7a by transactivating the expression of its primary transcripts. Consistent with previous reports that Dicer promotes EBV reactivation, we found that a let-7a mimic inhibited EBV reactivation to the lytic cycle, while a let-7 sponge increased reactivation. The results provide a mechanism by which EBNA1 could promote EBV latency by inducing let-7 miRNAs. IMPORTANCE: The EBNA1 protein of Epstein-Barr virus (EBV) contributes in multiple ways to the latent mode of EBV infection that leads to lifelong infection. In this study, we identify a mechanism by which EBNA1 helps to maintain EBV infection in a latent state. This involves induction of a family of microRNAs (let-7 miRNAs) that in turn decreases the level of the cellular protein Dicer. We demonstrate that let-7 miRNAs inhibit the reactivation of latent EBV, providing an explanation for our previous observation that EBNA1 promotes latency. In addition, since decreased levels of Dicer have been associated with metastatic potential, EBNA1 may increase metastases by downregulating Dicer.

A multitasking Argonaute: exploring the many facets of C. elegans CSR-1.

While initial studies of small RNA-mediated gene regulatory pathways focused on the cytoplasmic functions of such pathways, identifying roles for Argonaute/small RNA pathways in modulating chromatin and organizing the genome has become a topic of intense research in recent years. Nuclear regulatory mechanisms for Argonaute/small RNA pathways appear to be widespread, in organisms ranging from plants to fission yeast, Caenorhabditis elegans to humans. As the effectors of small RNA-mediated gene regulatory pathways, Argonaute proteins guide the chromatin-directed activities of these pathways. Of particular interest is the C. elegans Argonaute, chromosome segregation and RNAi deficient (CSR-1), which has been implicated in such diverse functions as organizing the holocentromeres of worm chromosomes, modulating germline chromatin, protecting the genome from foreign nucleic acid, regulating histone levels, executing RNAi, and inhibiting translation in conjunction with Pumilio proteins. CSR-1 interacts with small RNAs known as 22G-RNAs, which have complementarity to 25 % of the protein coding genes. This peculiar Argonaute is the only essential C. elegans Argonaute out of 24 family members in total. Here, we summarize the current understanding of CSR-1 functions in the worm, with emphasis on the chromatin-directed activities of this ever-intriguing Argonaute.

Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway.

Argonaute (AGO) proteins interact with distinct classes of small RNAs to direct multiple regulatory outcomes. In many organisms, including plants, fungi, and nematodes, cellular RNA-dependent RNA polymerases (RdRPs) use AGO targets as templates for amplification of silencing signals. Here, we show that distinct RdRPs function sequentially to produce small RNAs that target endogenous loci in Caenorhabditis elegans. We show that DCR-1, the RdRP RRF-3, and the dsRNA-binding protein RDE-4 are required for the biogenesis of 26-nt small RNAs with a 5' guanine (26G-RNAs) and that 26G-RNAs engage the Piwi-clade AGO, ERGO-1. Our findings support a model in which targeting by ERGO-1 recruits a second RdRP (RRF-1 or EGO-1), which in turn transcribes 22G-RNAs that interact with worm-specific AGOs (WAGOs) to direct gene silencing. ERGO-1 targets exhibit a nonrandom distribution in the genome and appear to include many gene duplications, suggesting that this pathway may control overexpression resulting from gene expansion.

Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans.

Gametogenesis is a thermosensitive process in numerous metazoans, ranging from worms to man. In Caenorhabditis elegans, a variety of RNA-binding proteins that associate with germ-line nuage (P granules), including the Piwi-clade argonaute PRG-1, have been implicated in maintaining fertility at elevated temperature. Here we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3), in promoting thermotolerant male fertility. A rescuing GFP::alg-3 transgene is localized to P granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, the 26G-RNAs which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for thermotolerant fertility in C. elegans and support a model in which AGOs and their small RNA cofactors function to promote robustness in gene-expression networks.

Analysis of C. elegans Germline Small RNA Pathways.

Sequence-specific gene regulation by small RNA (sRNA) pathways is essential for the development and function of organisms in all domains of life. These regulatory complexes, containing an Argonaute protein (AGO) guided by a bound sRNA, have the potential to regulate thousands of individual target transcripts at both the co- and post-transcriptional level. Determining the repertoire of transcripts that an AGO is capable of regulating in a particular context is essential to understanding the function of these regulatory modules. Immunoprecipitation (IP) of AGOs and subsequent RNA sequencing of their bound sRNAs allows for the inference of their target transcripts by mapping the sequences of the co-precipitated sRNAs back to their complementary target transcripts. This approach can be complemented by sequencing sRNAs from ago mutants as sRNA transcripts are degraded in the absence of their AGO binding partner. Here, we describe a framework for analyzing AGO/sRNA pathways in the germline, from using CRISPR-Cas9 to tag or mutate AGOs, through protocols for the extraction, sequencing, and analysis of sRNAs from AGO IPs and ago mutants.