A comprehensive survey of C. elegans argonaute proteins reveals organism-wide gene regulatory networks and functions.

Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode Caenorhabditis elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work, we systematically analyzed every C. elegans AGO using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss.

PIWI puts spermatogenesis in its place.

In this issue of Developmental Cell, Cornes et al. show that piRNAs initiate transcriptional silencing of spermatogenesis genes in the C. elegans germline via an endogenous nuclear RNAi pathway. This silencing enables a timely transition from spermatogenesis to oogenesis during hermaphrodite development, thus promoting fertility.

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.

A journey to 'tame a small metazoan organism', ‡ seen through the artistic eyes of C. elegans researchers.

In the following pages, we share a collection of photos, drawings, and mixed-media creations, most of them especially made for this JoN issue, manifesting C. elegans researchers' affection for their model organism and the founders of the field. This is a celebration of our community's growth, flourish, spread, and bright future. Descriptions provided by the contributors, edited for space. 1.

P Granules Protect RNA Interference Genes from Silencing by piRNAs.

P granules are perinuclear condensates in C. elegans germ cells proposed to serve as hubs for self/non-self RNA discrimination by Argonautes. We report that a mutant (meg-3 meg-4) that does not assemble P granules in primordial germ cells loses competence for RNA-interference over several generations and accumulates silencing small RNAs against hundreds of endogenous genes, including the RNA-interference genes rde-11 and sid-1. In wild type, rde-11 and sid-1 transcripts are heavily targeted by piRNAs and accumulate in P granules but maintain expression. In the primordial germ cells of meg-3 meg-4 mutants, rde-11 and sid-1 transcripts disperse in the cytoplasm with the small RNA biogenesis machinery, become hyper-targeted by secondary sRNAs, and are eventually silenced. Silencing requires the PIWI-class Argonaute PRG-1 and the nuclear Argonaute HRDE-1 that maintains trans-generational silencing of piRNA targets. These observations support a "safe harbor" model for P granules in protecting germline transcripts from piRNA-initiated silencing.

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.