A small nucleolar RNA functions in rRNA processing in Caenorhabditis elegans

CeR-2 RNA is one of the newly identified Caenorhabditis elegans noncoding RNAs (ncRNAs). The characterization of CeR-2 by RNomic studies has failed to classify it into any known ncRNA family. In this study, we examined the spatiotemporal expression patterns of CeR-2 to gain insight into its function. CeR-2 is expressed in most cells from the early embryo to adult stages. The subcellular localization of this RNA is analogous to that of fibrillarin, a major protein of the nucleolus. It was observed that knockdown of C/D small nucleolar ribonucleoproteins (snoRNPs), but not of H/ACA snoRNPs, resulted in the aberrant nucleolar localization of CeR-2 RNA. A mutant worm with a reduced amount of cellular CeR-2 RNA showed changes in its pre-rRNA processing pattern compared with that of the wild-type strain N2. These results suggest that CeR-2 RNA is a C/D snoRNA involved in the processing of rRNAs.     

Inducible systemic RNA silencing in Caenorhabditis elegans

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.     

Endogenous inhibitors of RNA interference in Caenorhabditis elegans

In eukaryotes, double-stranded RNAs (dsRNAs) or short, interfering dsRNAs (siRNAs) can reduce the accumulation of a sequence-related mRNA, often resulting in a loss-of-function phenotype-a process termed RNA interference (RNAi). Unfortunately, some mRNAs are resistant to the effects of dsRNA. Experiments designed to unravel RNAi mechanisms in Caenorhabditis elegans have led to the identification of two worm proteins, RRF-31,2 and, now, ERI-1,3 that can inhibit RNAi responses. Animals defective in either protein can display enhanced RNAi phenotypes for mRNAs that were previously resistant to dsRNA. Since ERI-1 is a conserved protein, development of procedures to enhance RNAi effectiveness in other systems may be possible.     

Structural analysis of N-linked glycans in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Man(alpha)1-6(Man(alpha)1-3)Man(beta)1-4GlcNAc(beta)1-4GlcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.     

Argonaute:The executor of small RNA function

The discovery ot small non-coding RNAs- microRNA(miRNA),short interfering RNA(siRNA) and PIWIinteracting RNA(piRNA)- represents one of the most exciting frontiers in biology specifically on the mechanism of gene regulation.In order to execute their functions,these small RNAs require physical interactions with their protein partners,the Argonaute(AGO) family proteins.Over the years,numerous studies have made tremendous progress on understanding the roles of AGO in gene silencing in various organisms.In this review,we summarize recent progress of AGO-mediated gene silencing and other cellular processes in which AGO proteins have been implicated with a particular focus on progress made in flies,humans and other model organisms as compliment.     

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.     

Structural insights into small RNA sorting and mRNA target binding by Arabidopsis Argonaute Mid domains

The RISC-associated Argonaute (Ago) proteins play the catalytic role for RISC-mediated gene regulation by selecting small RNAs and subsequent targeting and cleavage of complementary mRNAs. Ago Mid domains are proposed to play essential roles in small RNA sorting. Here, we report the crystal structures of Arabidopsis Ago1 Mid domain and its chimera mutant with part of Ago1 replaced by Ago4. The structures demonstrate that a single amino insertion in the nucleotide specificity loop of AtAgo1 will change the nucleotide binding preference of AtAgo1 from “5′-U” to “5′-A”. Moreover, we identify a long positively charged groove located along the “5′-end-nucleotide specificity loop” and occupied by several sulfate ions with the distance of 9-11 Å distance, indicating a putative mRNA target binding groove.     

Structural analysis of hyperperiodic DNA from Caenorhabditis elegans

Several bioinformatics studies have identified an unexpected but remarkably prevalent ~10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. In this work, we demonstrate that several hyperperiodic DNA segments from C.elegans exhibit structural anomalies using high-resolution atomic force microscopy (AFM) and gel electrophoresis. Our quantitative analysis of AFM images reveals that hyperperiodic DNA adopts a significantly smaller mean square end-to-end distance, hence a more compact coil structure, compared with non-periodic DNA of similar length. While molecules remain capable of adopting both bent and straight (rod-like) configurations, indicating that their flexibility is still retained, examination of the local curvatures along the DNA contour length reveals that the decreased mean square end-to-end distance can be attributed to the presence of long-scale intrinsic bending in hyperperiodic DNA. Such bending is not detected in non-periodic DNA. Similar studies of shorter, nucleosome-length DNAs that survived micrococcal nuclease digestion show that sequence hyperperiodicity in short segments can likewise induce strong intrinsic bending. It appears, therefore, that regions of the C.elegans genome display a significant correlation between DNA sequence and unusual mechanical properties.     

Structural properties of the Caenorhabditis elegans neuronal network

Despite recent interest in reconstructing neuronal networks, complete wiring diagrams on the level of individual synapses remain scarce and the insights into function they can provide remain unclear. Even for Caenorhabditis elegans, whose neuronal network is relatively small and stereotypical from animal to animal, published wiring diagrams are neither accurate nor complete and self-consistent. Using materials from White et al. and new electron micrographs we assemble whole, self-consistent gap junction and chemical synapse networks of hermaphrodite C. elegans. We propose a method to visualize the wiring diagram, which reflects network signal flow. We calculate statistical and topological properties of the network, such as degree distributions, synaptic multiplicities, and small-world properties, that help in understanding network signal propagation. We identify neurons that may play central roles in information processing, and network motifs that could serve as functional modules of the network. We explore propagation of neuronal activity in response to sensory or artificial stimulation using linear systems theory and find several activity patterns that could serve as substrates of previously described behaviors. Finally, we analyze the interaction between the gap junction and the chemical synapse networks. Since several statistical properties of the C. elegans network, such as multiplicity and motif distributions are similar to those found in mammalian neocortex, they likely point to general principles of neuronal networks. The wiring diagram reported here can help in understanding the mechanistic basis of behavior by generating predictions about future experiments involving genetic perturbations, laser ablations, or monitoring propagation of neuronal activity in response to stimulation.     

Overlapping stage-specific sets of numerous small collagenous polypeptides are translated in vitro from Caenorhabditis elegans RNA

Caenorhabditis elegans synthesizes four morphologically distinct types of collagenous cuticles during its lifetime. We show that in RNA populations isolated early or late during the L4-to-adult molt, chick and nematode collagen DNAs hybridize strongly to RNAs of about 1.2 kb. Different but overlapping classes of correspondingly small collagenous polypeptides (310-460 residues in length) are translated in vitro from these two populations and from RNA isolated at the L2-to-dauer molt. Over 60 different collagenous translation products are identified. These collagenous polypeptides are smaller than mature cuticle collagens and smaller than most vertebrate collagens. They probably represent cuticle collagen precursors and the primary products of the cuticle collagen genes of C. elegans.     

Uncover Genetic Interactions in Caenorhabditis elegans by RNA Interference

RNA-mediated interference (RNAi) has emerged recently as one of the most powerful functional genomics tools. RNAi has been particularly effective in the nematode worm C. elegans where RNAi has been used to analyse the loss-of-function phenotypes of almost all predicted genes. In this review, we illustrate how RNAi has been used to analyse gene function in C. elegans as well as pointing to some future directions for using RNAi to examine genetic interactions in a systematic manner.     

Phosphorylation of human Argonaute proteins affects small RNA binding

Argonaute (Ago) proteins are highly conserved between species and constitute a direct-binding platform for small RNAs including short-interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas Ago proteins are the actual mediators of gene silencing. Although the major steps in RNA-guided gene silencing have been elucidated, not much is known about Ago-protein regulation. Here we report a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small RNA 5'-end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate (E) mimicking a phosphorylated tyrosine, we show that small RNA binding is strongly reduced. Our data suggest that a negatively charged phospho-tyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5' phosphate of the small RNA.     

Genes required for systemic RNA interference in Caenorhabditis elegans

RNA interference (RNAi) in the nematode worm, Caenorhabditis elegans, occurs systemically. Double-stranded RNA (dsRNA) provided in the diet can be absorbed from the gut lumen and distributed throughout the body, triggering RNAi in tissues that are not exposed to the initial dsRNA trigger. This is in marked contrast to other animals, in which RNAi does not spread from targeted tissues to neighboring cells. Here, we report the characterization of mutants defective in the systemic aspect of RNAi, but not in the core RNAi process itself. Analysis of these mutants suggests that dsRNA uptake is a specific process involving several unique proteins.     

Gene silencing in Caenorhabditis elegans by transitive RNA interference

When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5' to 3' synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi.     

Protease-mediated processing of Argonaute proteins controls small RNA association

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Neural network features distinguish chemosensory stimuli in Caenorhabditis elegans

Nervous systems extract and process information from the environment to alter animal behavior and physiology. Despite progress in understanding how different stimuli are represented by changes in neuronal activity, less is known about how they affect broader neural network properties. We developed a framework for using graph-theoretic features of neural network activity to predict ecologically relevant stimulus properties, in particular stimulus identity. We used the transparent nematode, Caenorhabditis elegans, with its small nervous system to define neural network features associated with various chemosensory stimuli. We first immobilized animals using a microfluidic device and exposed their noses to chemical stimuli while monitoring changes in neural activity of more than 50 neurons in the head region. We found that graph-theoretic features, which capture patterns of interactions between neurons, are modulated by stimulus identity. Further, we show that a simple machine learning classifier trained using graph-theoretic features alone, or in combination with neural activity features, can accurately predict salt stimulus. Moreover, by focusing on putative causal interactions between neurons, the graph-theoretic features were almost twice as predictive as the neural activity features. These results reveal that stimulus identity modulates the broad, network-level organization of the nervous system, and that graph theory can be used to characterize these changes.     

Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing

Small RNAs loaded into Argonaute proteins direct silencing of complementary target mRNAs. It has been proposed that multiple, imperfectly complementary small interfering RNAs or microRNAs, when bound to the 3' untranslated region of a target mRNA, function cooperatively to silence target expression. We report that, in cultured human HeLa cells and mouse embryonic fibroblasts, Argonaute1 (Ago1), Ago3, and Ago4 act cooperatively to silence both perfectly and partially complementary target RNAs bearing multiple small RNA-binding sites. Our data suggest that for Ago1, Ago3, and Ago4, multiple, adjacent small RNA-binding sites facilitate cooperative interactions that stabilize Argonaute binding. In contrast, small RNAs bound to Ago2 and pairing perfectly to an mRNA target act independently to silence expression. Noncooperative silencing by Ago2 does not require the endoribonuclease activity of the protein: A mutant Ago2 that cannot cleave its mRNA target also silences noncooperatively. We propose that Ago2 binds its targets by a mechanism fundamentally distinct from that used by the three other mammalian Argonaute proteins.