Kato T et al. (2002) Glycobiology "Identification of an endo-beta-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli."

Kumagai H, Ikura K, Fujita K, Kato T, Natsuka S, Kobayashi K, Yamamoto K, Takeuchi M

[Glycobiology, 2002]

We report the identification, molecular cloning, and characterization of an endo-beta-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-beta-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-beta-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-beta-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man8GlcNAc2 was the best substrate for Endo-CE, and Man3GlcNAc2 was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed. Its substrate specificity was similar to those of Endo-M and endo-beta-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-beta-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.

Cohen-Berkman M et al. (2020) Elife "Endogenous siRNAs promote proteostasis and longevity in germline less C. elegans."

Dudkevich R, Lamm AT, Cohen-Berkman M, Henis-Korenblit S, Fishman A, Ben-Hamo S, Salzberg Y, Waldman Ben-Asher H

[Elife, 2020]

How lifespan and the rate of aging are set is a key problem in biology. Small RNAs are conserved molecules that impact diverse biological processes through the control of gene expression. However, in contrast to miRNAs, the role of endo-siRNAs in aging remains unexplored. Here, by combining deep sequencing and genomic and genetic approaches in <i>C. elegans</i>, we reveal an unprecedented role for endo-siRNA molecules in the maintenance of proteostasis and lifespan extension in germline-less animals. Furthermore, we identify an endo-siRNA-regulated tyrosine phosphatase, which limits the longevity of germline-less animals by restricting the activity of the heat shock transcription factor HSF-1. Altogether, our findings point to endo-siRNAs as a link between germline removal and the HSF-1 proteostasis and longevity-promoting somatic pathway. This establishes a role for endo siRNAs in the aging process and identifies downstream genes and physiological processes that are regulated by the endo siRNAs to affect longevity.

Pavelec DM et al. (2009) Genetics "Requirement for the ERI/DICER complex in endogenous RNA interference and sperm development in Caenorhabditis elegans."

Duchaine TF, Kennedy S, Smith HE, Pavelec DM, Lachowiec J

[Genetics, 2009]

Small regulatory RNAs are key regulators of gene expression. One class of small regulatory RNAs, termed the endogenous small interfering RNAs (endo siRNAs), is thought to negatively regulate cellular transcripts via an RNA interference (RNAi)-like mechanism termed endogenous RNAi (endo RNAi). A complex of proteins composed of ERI-1/3/5, RRF-3, and DICER (the ERI/DICER complex) mediates endo RNAi processes in Caenorhabditis elegans. We conducted a genetic screen to identify additional components of the endo RNAi machinery. Our screen recovered alleles of eri-9, which encodes a novel DICER-interacting protein, and a missense mutation within the helicase domain of DICER [DCR-1(G492R)]. ERI-9(-) and DCR-1(G492) animals exhibit defects in endo siRNA expression and a concomitant failure to regulate mRNAs that exhibit sequence homology to these endo siRNAs, indicating that ERI-9 and the DCR-1 helicase domain function in the C. elegans endo RNAi pathway. We define a subset of Eri mutant animals (including eri-1, rrf-3, eri-3, and dcr-1, but not eri-9 or ergo-1) that exhibit temperature-sensitive, sperm-specific sterility and defects in X chromosome segregation. Among these mutants we find multiple aberrations in sperm development beginning with cytokinesis and extending through terminal differentiation. These results identify novel components of the endo RNAi machinery, demonstrate differential requirements for the Eri factors in the sperm-producing germline, and begin to delineate the functional requirement for the ERI/DICER complex in sperm development.

Shiu PK et al. (2014) Methods Mol Biol "Assays for direct and indirect effects of C. elegans endo-siRNAs."

Hunter CP, Shiu PK, Zhuang JJ

[Methods Mol Biol, 2014]

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. Quantification of these small RNAs and their effect on target RNAs is a powerful tool for the analysis of RNAi; however, detection of small RNAs can be difficult due to their small size and relatively low abundance. Here, we describe the novel FirePlex assay for directly detecting endo-siRNA levels in bulk, as well as an optimized qPCR method for detecting the effect of endo-siRNAs on gene targets. Intriguingly, the loss of endo-siRNAs frequently results in enhanced experimental RNAi. Thus, we also present an optimized method to assess the indirect impact of endo-siRNAs on experimental RNAi efficiency.

Welker NC et al. (2010) RNA "Dicer's helicase domain is required for accumulation of some, but not all, C. elegans endogenous siRNAs."

Bass BL, Duchaine TF, Kennedy S, Welker NC, Pavelec DM, Nix DA

[RNA, 2010]

Years after the discovery that Dicer is a key enzyme in gene silencing, the role of its helicase domain remains enigmatic. Here we show that this domain is critical for accumulation of certain endogenous small interfering RNAs (endo-siRNAs) in Caenorhabditis elegans. The domain is required for the production of the direct products of Dicer, or primary endo-siRNAs, and consequently affects levels of downstream intermediates, the secondary endo-siRNAs. Consistent with the role of endo-siRNAs in silencing, their loss correlates with an increase in cognate mRNA levels. We find that the helicase domain of Dicer is not necessary for microRNA (miRNA) processing, or RNA interference following exposure to exogenous double-stranded RNA. Comparisons of wild-type and helicase-defective strains using deep-sequencing analyses show that the helicase domain is required by a subset of annotated endo-siRNAs, in particular, those associated with the slightly longer 26-nucleotide small RNA species containing a 5' guanosine.

Thivierge C et al. (2012) Nat Struct Mol Biol "Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi."

Duchaine TF, Flamand M, Mello CC, Wohlschlegel J, Vasale JJ, Makil N, Thivierge C, Conte D

[Nat Struct Mol Biol, 2012]

Endogenous RNA interference (endo-RNAi) pathways use a variety of mechanisms to generate siRNA and to mediate gene silencing. In Caenorhabditis elegans, DCR-1 is essential for competing RNAi pathways-the ERI endo-RNAi pathway and the exogenous RNAi pathway-to function. Here, we demonstrate that DCR-1 forms exclusive complexes in each pathway and further define the ERI-DCR-1 complex. We show that the tandem tudor protein ERI-5 potentiates ERI endo-RNAi by tethering an RNA-dependent RNA polymerase (RdRP) module to DCR-1. In the absence of ERI-5, the RdRP module is uncoupled from DCR-1. Notably, EKL-1, an ERI-5 paralog that specifies distinct RdRP modules in Dicer-independent endo-RNAi pathways, partially compensates for the loss of ERI-5 without interacting with DCR-1. Our results implicate tudor proteins in the recruitment of RdRP complexes to specific steps within DCR-1-dependent and DCR-1-independent endo-RNAi pathways.

Zhuang JJ et al. (2012) Genes (Basel) "The Influence of Competition Among C. elegans Small RNA Pathways on Development."

Zhuang JJ, Hunter CP

[Genes (Basel), 2012]

Small RNAs play a variety of regulatory roles, including highly conserved developmental functions. Caenorhabditis elegans not only possesses most known small RNA pathways, it is also an easy system to study their roles and interactions during development. It has been proposed that in C. elegans, some small RNA pathways compete for access to common limiting resources. The strongest evidence supporting this model is that disrupting the production or stability of endogenous short interfering RNAs (endo-siRNAs) enhances sensitivity to experimentally induced exogenous RNA interference (exo-RNAi). Here, we examine the relationship between the endo-siRNA and microRNA (miRNA) pathways, and find that, consistent with competition among these endogenous small RNA pathways, endo-siRNA pathway mutants may enhance miRNA efficacy. Furthermore, we show that exo-RNAi may also compete with both endo-siRNAs and miRNAs. Our data thus provide support that all known Dicer-dependent small RNA pathways may compete for limiting common resources. Finally, we observed that both endo-siRNA mutants and animals experiencing exo-RNAi have increased expression of miRNA-regulated stage-specific developmental genes. These observations suggest that perturbing the small RNA flux and/or the induction of exo-RNAi, even in wild-type animals, may impact development via effects on the endo-RNAi and microRNA pathways.

Elewa A et al. (2015) Dev Cell "POS-1 Promotes Endo-mesoderm Development by Inhibiting the Cytoplasmic Polyadenylation of neg-1 mRNA."

Harrison PF, Du Z, Woolf H, Ishidate T, Elewa A, Powell DR, Beilharz TH, Ryder SP, Kaymak E, Bao Z, Yi D, Shirayama M, Mello CC, Srinivasan J, Chute CD

[Dev Cell, 2015]

The regulation of mRNA translation is of fundamental importance in biological mechanisms ranging from embryonic axis specification to the formation of long-term memory. POS-1 is one of several CCCH zinc-finger RNA-binding proteins that regulate cell fate specification during C. elegans embryogenesis. Paradoxically, pos-1 mutants exhibit striking defects in endo-mesoderm development but have wild-type distributions of SKN-1, a key determinant of endo-mesoderm fates. RNAi screens for pos-1 suppressors identified genes encoding the cytoplasmic poly(A)-polymerase homolog GLD-2, the Bicaudal-C homolog GLD-3, and the protein NEG-1. We show that NEG-1 localizes in anterior nuclei, where it negatively regulates endo-mesoderm fates. In posterior cells, POS-1 binds the neg-1 3' UTR to oppose GLD-2 and GLD-3 activities that promote NEG-1 expression and cytoplasmic lengthening of the neg-1 mRNA poly(A) tail. Our findings uncover an intricate series of post-transcriptional regulatory interactions that, together, achieve precise spatial expression of endo-mesoderm fates in C. elegans embryos.

Han T et al. (2009) Proc Natl Acad Sci U S A "26G endo-siRNAs regulate spermatogenic and zygotic gene expression in Caenorhabditis elegans."

Chu DS, Bouffard P, Thierry-Mieg J, Harkins TT, Manoharan AP, Fitzpatrick C, Han T, Thierry-Mieg D, Kim JK

[Proc Natl Acad Sci U S A, 2009]

Endogenous small interfering RNAs (endo-siRNAs) regulate diverse gene expression programs in eukaryotes by either binding and cleaving mRNA targets or mediating heterochromatin formation; however, the mechanisms of endo-siRNA biogenesis, sorting, and target regulation remain poorly understood. Here we report the identification and function of a specific class of germline-generated endo-siRNAs in Caenorhabditis elegans that are 26 nt in length and contain a guanine at the first nucleotide position (i.e., 26G RNAs). 26G RNAs regulate gene expression during spermatogenesis and zygotic development, and their biogenesis requires the ERI-1 exonuclease and the RRF-3 RNA-dependent RNA polymerase (RdRP). Remarkably, we identified two nonoverlapping subclasses of 26G RNAs that sort into specific RNA-induced silencing complexes (RISCs) and differentially regulate distinct mRNA targets. Class I 26G RNAs target genes are expressed during spermatogenesis, whereas class II 26G RNAs are maternally inherited and silence gene expression during zygotic development. These findings implicate a class of endo-siRNAs in the global regulation of transcriptional programs required for fertility and development.

Gu W et al. (2009) Mol Cell "Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline."

Shirayama M, Yates JR, Gu W, Vasale J, Duan S, Chaves DA, Claycomb JM, Mitani S, Mello CC, Keys J, Youngman EM, Moresco JJ, Chen CC, Conte D, Falgren N, Kasschau KD, Stoltz MJ, Carrington JC, Batista PJ

[Mol Cell, 2009]

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.