Explore the vast range of titles available.

In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus , which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species. Mammalian cell-derived exosomes can carry RNA and proteins from cell to cell, but this mode of transport has not been shown in nematodes. Here the authors show that a gastrointestinal parasite secretes exosomes that transfer microRNAs to mammalian cells and regulate innate immunity.

We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor-binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor-binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

NRDE-2 active in transcription regulation Small RNAs function in both the cytoplasm, inhibiting expression from messenger RNAs, and in the nucleus, to silence heterochromatin and prevent genome rearrangement. In this study, Guang et al . characterize a new protein involved in RNA interference in the nucleus. NRDE-2 associates with the Argonaute protein NRDE-3 and siRNAs on nascent transcripts. This association prevents elongation by RNA polymerase II, thereby making this a co-transcriptional form of gene silencing. Small regulatory RNAs function both in the cytoplasm, inhibiting expression from messenger RNAs, and in the nucleus, silencing heterochromatin and preventing genome rearrangement. Now a new protein involved in RNA interference in the nucleus has been characterized. This protein, NRDE-2, associates with NRDE-3 and short interfering RNAs on nascent transcripts. This association prevents elongation of the transcripts by RNA polymerase II, making this a co-transcriptional form of gene silencing. Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defence against parasitic nucleic acids and genome rearrangement. Many small regulatory RNAs are thought to function in nuclei 1 , 2 . For instance, in plants and fungi, short interfering RNA (siRNAs) associate with nascent transcripts and direct chromatin and/or DNA modifications 1 , 2 . To understand further the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in Caenorhabditis elegans nuclei 3 . Here we show that the gene nuclear RNAi defective-2 ( nrde-2 ) encodes an evolutionarily conserved protein that is required for siRNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct an NRDE-2-dependent silencing of pre-messenger RNAs (pre-mRNAs) 3′ to sites of RNAi, an NRDE-2-dependent accumulation of RNA polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3′ to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation: siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.

By silencing transposons, Piwi-interacting RNAs (piRNAs) protect the stability of animal genomes in germ lines. However, many piRNAs do not map to transposons, and their functions have remained undefined. Zhang et al. described the piRNA targeting logic in Caenorhabditis elegans and identified an intrinsic sequence signal in endogenous germline genes that confer resistance to piRNA silencing. Thus, diverse piRNAs silence foreign nucleic acids but spare self genes to defend the C. elegans genome. In addition, multiple foreign transgenes can be engineered to escape piRNA targeting, allowing successful expression in the germline. Science , this issue p. 587 In nematodes, endogenous genes are resistant to Piwi-interacting RNA (piRNA) silencing, allowing a role for piRNA in defense against foreign nucleic acids. Piwi-interacting RNAs (piRNAs) silence transposons to safeguard genome integrity in animals. However, the functions of the many piRNAs that do not map to transposons remain unknown. Here, we show that piRNA targeting in Caenorhabditis elegans can tolerate a few mismatches but prefer perfect pairing at the seed region. The broad targeting capacity of piRNAs underlies the germline silencing of transgenes in C. elegans . Transgenes engineered to avoid piRNA recognition are stably expressed. Many endogenous germline-expressed genes also contain predicted piRNA targeting sites, and periodic An/Tn clusters (PATCs) are an intrinsic signal that provides resistance to piRNA silencing. Together, our study revealed the piRNA targeting rules and highlights a distinct strategy that C. elegans uses to distinguish endogenous from foreign nucleic acids.

Ribonucleoprotein complexes consisting of Argonaute-like proteins and small regulatory RNAs function in a wide range of biological processes. Many of these small regulatory RNAs are predicted to act, at least in part, within the nucleus. We conducted a genetic screen to identify factors essential for RNA interference (RNAi) in nuclei of Caenorhabditis elegans and identified the Argonaute protein NRDE-3. In the absence of small interfering RNAs (siRNAs), NRDE-3 resides in the cytoplasm. NRDE-3 binds siRNAs generated by RNA-dependent RNA polymerases acting on messenger RNA templates in the cytoplasm and redistributes to the nucleus. Nuclear redistribution of NRDE-3 requires a functional nuclear localization signal, is required for nuclear RNAi, and results in NRDE-3 association with nuclear-localized nascent transcripts. Thus, specific Argonaute proteins can transport specific classes of small regulatory RNAs to distinct cellular compartments to regulate gene expression.

RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: \"Primary siRNAs\" (derived from DICER nuclease-mediated cleavage of the original trigger) and \"secondary siRNAs\" [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.

RNA granules have been likened to liquid droplets whose dynamics depend on the controlled dissolution and condensation of internal components. The molecules and reactions that drive these dynamics in vivo are not well understood. In this study, we present evidence that a group of intrinsically disordered, serine-rich proteins regulate the dynamics of P granules in C. elegans embryos. The MEG (maternal-effect germline defective) proteins are germ plasm components that are required redundantly for fertility. We demonstrate that MEG-1 and MEG-3 are substrates of the kinase MBK-2/DYRK and the phosphatase PP2APPTR−½. Phosphorylation of the MEGs promotes granule disassembly and dephosphorylation promotes granule assembly. Using lattice light sheet microscopy on live embryos, we show that GFP-tagged MEG-3 localizes to a dynamic domain that surrounds and penetrates each granule. We conclude that, despite their liquid-like behavior, P granules are non-homogeneous structures whose assembly in embryos is regulated by phosphorylation. For a gene to be expressed as a protein, its DNA is first used as a template to make a molecule of RNA, which is then translated to make the protein. In most cells, RNA molecules concentrate into aggregates called RNA granules. These granules contain both RNA and proteins that bind to RNA and are used to transport, store, and regulate the translation and breakdown of RNA molecules. Unlike many other structures within cells, RNA granules are not surrounded by a membrane; and the molecules that hold RNA granules together are not known. P granules are a type of RNA granule that is found in the germ cells (the cells that go on to form eggs and sperm) of a microscopic worm called C. elegans. When a C. elegans embryo is still a single cell, P granules move throughout the cell and the P granules at the front of the cell dissolve, while those at the back condense. As such, when the single-celled embryo divides, the front forms a cell without P granules (that will go on to form the tissues of the worm's body) and the back becomes a P granule-containing germ cell. Two proteins called MBK-2 and PPTR-1 have opposite effects on P granules: MBK-2 causes P granules to dissolve, while PPTR-1 makes them form. MBK-2 is an enzyme that adds phosphate groups onto other proteins, whereas PPTR-1 is part of an enzyme that removes such groups. Wang et al. have now searched for proteins that interact with MBK-2 and PPTR-1 in order to identify the molecules that regulate the assembly of P granules. They found that a group of proteins, known as MEG proteins, are acted upon by both of these proteins. Wang et al. found that MBK-2 adds phosphate groups to MEG proteins, which encourages granules to disassemble, while PPTR-1 removes these groups to promote granule assembly. Wang et al. generated mutant worms that lacked each of the MEG proteins. These mutant worms had fewer and smaller P granules than normal worms. Without MEG proteins, P granules failed to assemble or disassemble normally and the worms were infertile. Using high resolution microscopy, Wang et al. observed that the MEG proteins wrap around the P granules and that one of the MEG proteins—called MEG-3—follows an almost ribbon-like path that surrounds and enters each granule. These observations suggest that the MEG proteins stabilize RNA granules by forming a cage-like scaffold around each granule. How the MEG proteins—which are predicted to lack a fixed or ordered three-dimensional structure and show no similarity to proteins with known functions—assemble into a scaffold will be the focus of future studies.

The phenotypic differences between individual organisms can often be ascribed to underlying genetic and environmental variation. However, even genetically identical organisms in homogeneous environments vary, indicating that randomness in developmental processes such as gene expression may also generate diversity. To examine the consequences of gene expression variability in multicellular organisms, we studied intestinal specification in the nematode Caenorhabditis elegans in which wild-type cell fate is invariant and controlled by a small transcriptional network. Mutations in elements of this network can have indeterminate effects: some mutant embryos fail to develop intestinal cells, whereas others produce intestinal precursors. By counting transcripts of the genes in this network in individual embryos, we show that the expression of an otherwise redundant gene becomes highly variable in the mutants and that this variation is subjected to a threshold, producing an ON/OFF expression pattern of the master regulatory gene of intestinal differentiation. Our results demonstrate that mutations in developmental networks can expose otherwise buffered stochastic variability in gene expression, leading to pronounced phenotypic variation. Gene expression differences In 1925 Timoféeff-Ressovsky and Romaschoff independently noticed that individuals harbouring identical mutant alleles can show either mutant or wild-type phenotypes, a property known as incomplete penetrance. It has since emerged that individual organisms that are genetically identical can display different characteristics even in identical environments, and that there is an element of randomness in gene expression. In recent years such 'noisy' gene expression has been studied quantitatively in microbes. Now Alexander van Oudenaarden and colleagues extend the quantitative approach to a multicellular organism, the Caenorhabditis elegans roundworm. Using single mRNA molecule counting they show how mutations in genes regulating the production of intestinal cells in the embryo lead to higher randomness in other gene levels and in resulting phenotypes, despite identical genotypes and fixed environmental conditions. The work also suggests how gene regulatory networks may evolve robustness to individual mutations. Even genetically identical organisms in homogeneous environments vary, indicating that randomness in developmental processes such as gene expression may generate phenotypic diversity. Intestinal specification in the nematode Caenorhabditis elegans , in which wild-type cell fate is invariant and controlled by a small transcriptional network, is now studied. The results demonstrate that mutations in developmental networks can expose stochastic variability in gene expression, leading to phenotypic variation.

Schistosomiasis is one of the most prevalent parasitic diseases, affecting millions of people in developing countries. Amongst the human-infective species, Schistosoma mansoni is also the most commonly used in the laboratory and here we present the systematic improvement of its draft genome. We used Sanger capillary and deep-coverage Illumina sequencing from clonal worms to upgrade the highly fragmented draft 380 Mb genome to one with only 885 scaffolds and more than 81% of the bases organised into chromosomes. We have also used transcriptome sequencing (RNA-seq) from four time points in the parasite's life cycle to refine gene predictions and profile their expression. More than 45% of predicted genes have been extensively modified and the total number has been reduced from 11,807 to 10,852. Using the new version of the genome, we identified trans-splicing events occurring in at least 11% of genes and identified clear cases where it is used to resolve polycistronic transcripts. We have produced a high-resolution map of temporal changes in expression for 9,535 genes, covering an unprecedented dynamic range for this organism. All of these data have been consolidated into a searchable format within the GeneDB (www.genedb.org) and SchistoDB (www.schistodb.net) databases. With further transcriptional profiling and genome sequencing increasingly accessible, the upgraded genome will form a fundamental dataset to underpin further advances in schistosome research.

Post-transcriptional gene regulation frequently occurs through elements in mRNA 3' untranslated regions (UTRs). Although crucial roles for 3'UTR-mediated gene regulation have been found in Caenorhabditis elegans, most C. elegans genes have lacked annotated 3'UTRs. Here we describe a high-throughput method for reliable identification of polyadenylated RNA termini, and we apply this method, called poly(A)-position profiling by sequencing (3P-Seq), to determine C. elegans 3'UTRs. Compared to standard methods also recently applied to C. elegans UTRs, 3P-Seq identified 8,580 additional UTRs while excluding thousands of shorter UTR isoforms that do not seem to be authentic. Analysis of this expanded and corrected data set suggested that the high A/U content of C. elegans 3'UTRs facilitated genome compaction, because the elements specifying cleavage and polyadenylation, which are A/U rich, can more readily emerge in A/U-rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,480 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes use palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3'UTRs have median length only one-sixth that of mammalian 3'UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying post-transcriptional gene regulation.