SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

Stable Heritable Germline Silencing Directs Somatic Silencing at an Endogenous Locus.

The importance of transgenerationally inherited epigenetic states to organismal fitness remains unknown as well-documented examples are often not amenable to mechanistic analysis or rely on artificial reporter loci. Here we describe an induced silenced state at an endogenous locus that persists, at 100% transmission without selection, for up to 13 generations. This unusually persistent silencing enables a detailed molecular genetic analysis of an inherited epigenetic state. We find that silencing is dependent on germline nuclear RNAi factors and post-transcriptional mechanisms. Consistent with these later observations, inheritance does not require the silenced locus, and we provide genetic evidence that small RNAs embody the inherited silencing signal. Notably, heritable germline silencing directs somatic epigenetic silencing. Somatic silencing does not require somatic nuclear RNAi but instead requires both maternal germline nuclear RNAi and chromatin-modifying activity. Coupling inherited germline silencing to somatic silencing may enable selection for physiologically important traits.

Intergenerational Transmission of Gene Regulatory Information in Caenorhabditis elegans.

Epigenetic mechanisms can stably maintain gene expression states even after the initiating conditions have changed. Often epigenetic information is transmitted only to daughter cells, but evidence is emerging, in both vertebrate and invertebrate systems, for transgenerational epigenetic inheritance (TEI), the transmission of epigenetic gene regulatory information across generations. Each new description of TEI helps uncover the properties, molecular mechanisms and biological roles for TEI. The nematode Caenorhabditis elegans has been particularly instrumental in the effort to understand TEI, as multiple environmental and genetic triggers can initiate an epigenetic signal that can alter the expression of both transgenes and endogenous loci. Here, we review recent studies of TEI in C. elegans.

Uptake of extracellular double-stranded RNA by SID-2.

Ingested dsRNAs trigger RNA interference (RNAi) in many invertebrates, including the nematode Caenorhabditis elegans. Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C. elegans for the import of ingested dsRNA and that, when expressed in Drosophila S2 cells, SID-2 enables the uptake of dsRNAs. SID-2-dependent dsRNA transport requires an acidic extracellular environment and is selective for dsRNAs with at least 50 base pairs. Through structure-function analysis, we identify several SID-2 regions required for this activity, including three extracellular, positively charged histidines. Finally, we find that SID-2-dependent transport is inhibited by drugs that interfere with vesicle transport. Therefore, we propose that environmental dsRNAs are imported from the acidic intestinal lumen by SID-2 via endocytosis and are released from internalized vesicles in a secondary step mediated by the dsRNA channel SID-1. Similar multistep mechanisms may underlie the widespread observations of environmental RNAi.

Conserved tyrosine kinase promotes the import of silencing RNA into Caenorhabditis elegans cells.

RNA silencing in Caenorhabditis elegans is transmitted between cells by the transport of double-stranded RNA (dsRNA). The efficiency of such transmission, however, depends on both the cell type and the environment. Here, we identify systemic RNAi defective-3 (SID-3) as a conserved tyrosine kinase required for the efficient import of dsRNA. Without SID-3, cells perform RNA silencing well but import dsRNA poorly. Upon overexpression of SID-3, cells import dsRNA more efficiently than do wild-type cells and such efficient import of dsRNA requires an intact SID-3 kinase domain. The mammalian homolog of SID-3, activated cdc-42-associated kinase (ACK), acts in many signaling pathways that respond to environmental changes and is known to directly associate with endocytic vesicles, which have been implicated in dsRNA transport. Therefore, our results suggest that the SID-3/ACK tyrosine kinase acts as a regulator of RNA import into animal cells.

SID-1 is a dsRNA-selective dsRNA-gated channel.

Systemic RNAi in Caenorhabditis elegans requires the widely conserved transmembrane protein SID-1 to transport RNAi silencing signals between cells. When expressed in Drosophila S2 cells, C. elegans SID-1 enables passive dsRNA uptake from the culture medium, suggesting that SID-1 functions as a channel for the transport of double-stranded RNA (dsRNA). Here we show that nucleic acid transport by SID-1 is specific for dsRNA and that addition of dsRNA to SID-1 expressing cells results in changes in membrane conductance, which indicate that SID-1 is a dsRNA gated channel protein. Consistent with passive bidirectional transport, we find that the RNA induced silencing complex (RISC) is required to prevent the export of imported dsRNA and that retention of dsRNA by RISC does not seem to involve processing of retained dsRNA into siRNAs. Finally, we show that mimics of natural molecules that contain both single- and double-stranded dsRNA, such as hairpin RNA and pre-microRNA, can be transported by SID-1. These findings provide insight into the nature of potential endogenous RNA signaling molecules in animals.

A genomic bias for genotype-environment interactions in C. elegans.

The phenotype of an organism is determined by its genotype and environment. An interaction between these two arises from the differential effect of the environment on gene expression in distinct genotypes; however, the genomic properties identifying these are not well understood. Here we analyze the transcriptomes of five C. elegans strains (genotype) cultivated in five growth conditions (environment), and find that highly regulated genes, as distinguished by intergenic lengths, motif concentration, and expression levels, are particularly biased toward genotype-environment interactions. Sequencing these strains, we find that genes with expression variation across genotypes are enriched for promoter single-nucleotide polymorphisms (SNPs), as expected. However, genes with genotype-environment interactions do not significantly differ from background in terms of their promoter SNPs. Collectively, these results indicate that the highly regulated nature of particular genes predispose them for exhibiting genotype-environment interaction as a consequence of changes to upstream regulators. This observation may provide a deeper understanding into the origin of the extraordinary gene expression diversity present in even closely related species.

Comparison of diverse developmental transcriptomes reveals that coexpression of gene neighbors is not evolutionarily conserved.

Genomic analyses have shown that adjacent genes are often coexpressed. However, it remains unclear whether the observed coexpression is a result of functional organization or a consequence of adjacent active chromatin or transcriptional read-through, which may be free of selective biases. Here, we compare temporal expression profiles of one-to-one orthologs in conserved or divergent genomic positions in two genetically distant nematode species-Caenorhabditis elegans and C. briggsae-that share a near-identical developmental program. We find, for all major patterns of temporal expression, a substantive amount of gene expression divergence. However, this divergence is not random: Genes that function in essential developmental processes show less divergence than genes whose functions are not required for viability. Coexpression of gene neighbors in either species is highly divergent in the other, in particular when the neighborhood is not conserved. Interestingly, essential genes appear to maintain their expression profiles despite changes in neighborhoods suggesting exposure to stronger selection. Our results suggest that a significant fraction of the coexpression observed among gene neighbors may be accounted for by neutral processes, and further that these may be distinguished by comparative gene expression analyses.

RNA interference in Caenorhabditis elegans: uptake, mechanism, and regulation.

RNA interference (RNAi) is a powerful research tool that has enabled molecular insights into gene activity, pathway analysis, partial loss-of-function phenotypes, and large-scale genomic discovery of gene function. While RNAi works extremely well in the non-parasitic nematode C. elegans, it is also especially useful in organisms that lack facile genetic analysis. Extensive genetic analysis of the mechanisms, delivery and regulation of RNAi in C. elegans has provided mechanistic and phenomenological insights into why RNAi is so effective in this species. These insights are useful for the testing and development of RNAi in other nematodes, including parasitic nematodes where more effective RNAi would be extremely useful. Here, we review the current advances in C. elegans for RNA delivery methods, regulation of cell autonomous and systemic RNAi phenomena, and implications of enhanced RNAi mutants. These discussions, with a focus on mechanism and cross-species application, provide new perspectives for optimizing RNAi in other species.

Export of RNA silencing from C. elegans tissues does not require the RNA channel SID-1.

Double-stranded RNA (dsRNA) triggers RNA interference (RNAi) to silence genes of matching sequence. In some animals this experimentally induced silencing is transported between cells, and studies in the nematode Caenorhabditis elegans have shown that the dsRNA channel SID-1 is required for the import of such transported silencing signals. Gene silencing can also be triggered by endogenously expressed RNAi triggers, but it is unknown whether such silencing is transported between cells. Here, we show that, in C. elegans, SID-1 is required for efficient silencing of multicopy transgenes, indicating that mobile silencing signals contribute to transgene silencing. Further, most tissues can transport silencing initiated by the tissue-specific transgenic expression of RNAi triggers to other tissues, consistent with expressed RNAi triggers generating mobile silencing signals. Whereas the import of silencing signals requires SID-1, we found that mobile silencing signals generated by transgene-expressed RNAi triggers are exported to other tissues through a SID-1-independent mechanism. Furthermore, when RNAi triggers are expressed in ingested Escherichia coli, silencing signals can be transported to internal tissues from the gut lumen across gut cells that lack SID-1. Thus, C. elegans can transport endogenous and exogenous RNA silencing signals between many different tissues via at least 2 SID-1 independent export pathways.

Cyclic tensile strain triggers a sequence of autocrine and paracrine signaling to regulate angiogenic sprouting in human vascular cells.

Mechanical signals regulate blood vessel development in vivo, and have been demonstrated to regulate signal transduction of endothelial cell (EC) and smooth muscle cell (SMC) phenotype in vitro. However, it is unclear how the complex process of angiogenesis, which involves multiple cell types and growth factors that act in a spatiotemporally regulated manner, is triggered by a mechanical input. Here, we describe a mechanism for modulating vascular cells during sequential stages of an in vitro model of early angiogenesis by applying cyclic tensile strain. Cyclic strain of human umbilical vein (HUV)ECs up-regulated the secretion of angiopoietin (Ang)-2 and PDGF-betabeta, and enhanced endothelial migration and sprout formation, whereas effects were eliminated with shRNA knockdown of endogenous Ang-2. Applying strain to colonies of HUVEC, cocultured on the same micropatterned substrate with nonstrained human aortic (HA)SMCs, led to a directed migration of the HASMC toward migrating HUVECs, with diminished recruitment when PDGF receptors were neutralized. These results demonstrate that a singular mechanical cue (cyclic tensile strain) can trigger a cascade of autocrine and paracrine signaling events between ECs and SMCs critical to the angiogenic process.

SID-4/NCK-1 is important for dsRNA import in Caenorhabditis elegans.

RNA interference is sequence-specific gene silencing triggered by double-stranded RNA. Systemic RNA interference is where double-stranded RNA, expressed or introduced into 1 cell, is transported to and initiates RNA interference in other cells. Systemic RNA interference is very efficient in Caenorhabditis elegans and genetic screens for systemic RNA interference-defective mutants have identified RNA transporters (SID-1, SID-2, and SID-5) and a signaling protein (SID-3). Here, we report that SID-4 is nck-1, a C. elegans NCK-like adaptor protein. sid-4 null mutations cause a weak, dose-sensitive, systemic RNA interference defect and can be effectively rescued by SID-4 expression in target tissues only, implying a role in double-stranded RNA import. SID-4 and SID-3 (ACK-1 kinase) homologs interact in mammals and insects, suggesting that they may function in a common signaling pathway; however, a sid-3; sid-4 double mutants showed additive resistance to RNA interference, suggesting that these proteins likely interact with other signaling pathways as well. A bioinformatic screen coupled to RNA interference sensitivity tests identified 23 additional signaling components with weak RNA interference-defective phenotypes. These observations suggest that environmental conditions may modulate systemic RNA interference efficacy, and indeed, sid-3 and sid-4 are required for growth temperature effects on systemic RNA interference silencing efficiency.

Environmental RNA interference.

The discovery of RNA interference (RNAi), the process of sequence-specific gene silencing initiated by double-stranded RNA (dsRNA), has broadened our understanding of gene regulation and has revolutionized methods for genetic analysis. A remarkable property of RNAi in the nematode Caenorhabditis elegans and in some other multicellular organisms is its systemic nature: silencing signals can cross cellular boundaries and spread between cells and tissues. Furthermore, C. elegans and some other organisms can also perform environmental RNAi: sequence-specific gene silencing in response to environmentally encountered dsRNA. This phenomenon has facilitated significant technological advances in diverse fields including functional genomics and agricultural pest control. Here, we describe the characterization and current understanding of environmental RNAi and discuss its potential applications.

The SID-1 double-stranded RNA transporter is not selective for dsRNA length.

The double-stranded RNA (dsRNA) transport protein SID-1 enables systemic RNA interference (RNAi) in Caenorhabditis elegans, whereby silencing initiated by local exposure to dsRNA spreads throughout the animal and to its progeny. Previously, we showed that providing dsRNA in the growth medium of Drosophila S2 cells that express C. elegans SID-1 efficiently triggers RNAi. In these experiments long dsRNA proved to be significantly more effective than short dsRNA in silencing the target gene. Here, we show that equivalent masses of long or short dsRNA accumulate in these cells, indicating that size-dependent silencing is not due to size-selective transport through SID-1. Furthermore, using pulse-chase dsRNA uptake experiments, we show that short dsRNA accumulates more rapidly than long dsRNA. We found that import rates are dependent on dsRNA concentration, consistent with energy-independent, diffusion-limited transport through the SID-1 channel. Comparison of silencing efficiencies between Drosophila S2 cells heterologously expressing SID-1 and primary-cultured C. elegans cells shows similar dsRNA concentration and size dependencies, suggesting that C. elegans regulatory proteins do not measurably enhance or restrict dsRNA transport through SID-1. Finally, we find that coexpressing mutant SID-1 with wild-type SID-1 in S2 cells interferes with SID-1 function, indicating that SID-1 may function as a multimer.

Pairing of competitive and topologically distinct regulatory modules enhances patterned gene expression.

Biological networks are inherently modular, yet little is known about how modules are assembled to enable coordinated and complex functions. We used RNAi and time series, whole-genome microarray analyses to systematically perturb and characterize components of a Caenorhabditis elegans lineage-specific transcriptional regulatory network. These data are supported by selected reporter gene analyses and comprehensive yeast one-hybrid and promoter sequence analyses. Based on these results, we define and characterize two modules composed of muscle- and epidermal-specifying transcription factors that function together within a single cell lineage to robustly specify multiple cell types. The expression of these two modules, although positively regulated by a common factor, is reliably segregated among daughter cells. Our analyses indicate that these modules repress each other, and we propose that this cross-inhibition coupled with their relative time of induction function to enhance the initial asymmetry in their expression patterns, thus leading to the observed invariant gene expression patterns and cell lineage. The coupling of asynchronous and topologically distinct modules may be a general principle of module assembly that functions to potentiate genetic switches.

Efficient Homologous Recombination in Mice Using Long Single Stranded DNA and CRISPR Cas9 Nickase.

The CRISPR/Cas9 nickase mutant is less prone to off-target double-strand (ds)DNA breaks than wild-type Cas9 because to produce dsDNA cleavage it requires two guide RNAs to target the nickase to nearby opposing strands. Like wild-type Cas9 lesions, these staggered lesions are repaired by either non-homologous end joining or, if a repair template is provided, by homologous recombination (HR). Here, we report very efficient (up to 100%) recovery of heterozygous insertions in Mus musculus produced by long (>300 nt), single-stranded DNA donor template-guided repair of paired-nickase lesions.

Semi-supervised analysis of gene expression profiles for lineage-specific development in the Caenorhabditis elegans embryo.

MOTIVATION: Gene expression profiling is a powerful approach to identify genes that may be involved in a specific biological process on a global scale. For example, gene expression profiling of mutant animals that lack or contain an excess of certain cell types is a common way to identify genes that are important for the development and maintenance of given cell types. However, it is difficult for traditional computational methods, including unsupervised and supervised learning methods, to detect relevant genes from a large collection of expression profiles with high sensitivity and specificity. Unsupervised methods group similar gene expressions together while ignoring important prior biological knowledge. Supervised methods utilize training data from prior biological knowledge to classify gene expression. However, for many biological problems, little prior knowledge is available, which limits the prediction performance of most supervised methods. RESULTS: We present a Bayesian semi-supervised learning method, called BGEN, that improves upon supervised and unsupervised methods by both capturing relevant expression profiles and using prior biological knowledge from literature and experimental validation. Unlike currently available semi-supervised learning methods, this new method trains a kernel classifier based on labeled and unlabeled gene expression examples. The semi-supervised trained classifier can then be used to efficiently classify the remaining genes in the dataset. Moreover, we model the confidence of microarray probes and probabilistically combine multiple probe predictions into gene predictions. We apply BGEN to identify genes involved in the development of a specific cell lineage in the C. elegans embryo, and to further identify the tissues in which these genes are enriched. Compared to K-means clustering and SVM classification, BGEN achieves higher sensitivity and specificity. We confirm certain predictions by biological experiments. AVAILABILITY: The results are available at http://www.csail.mit.edu/~alanqi/projects/BGEN.html.

Mutations in a beta-tubulin disrupt spindle orientation and microtubule dynamics in the early Caenorhabditis elegans embryo.

The early Caenorhabditis elegans embryo contains abundant transcripts for two alpha- and two beta-tubulins, raising the question of whether each isoform performs specialized functions or simply contributes to total tubulin levels. Our identification of two recessive, complementing alleles of a beta-tubulin that disrupt nuclear-centrosome centration and rotation in the early embryo originally suggested that this tubulin, tbb-2, has specialized functions. However, embryos from tbb-2 deletion worms do not have defects in nuclear-centrosome centration and rotation suggesting that the complementing alleles are not null mutations. Both complementing alleles have distinct effects on microtubule dynamics and show allele-specific interactions with the two embryonically expressed alpha-tubulins: One of the alleles causes microtubules to be cold stable and resistant to the microtubule-depolymerizing drug benomyl, whereas the other causes cell cycle-specific defects in microtubule polymerization. Gene-specific RNA interference targeting all four embryonically expressed tubulin genes singly and in all double combinations showed that the tubulin isoforms in the early embryo are largely functionally redundant with the exception of tbb-2. tbb-2 is required for centrosome stabilization during anaphase of the first cell division, suggesting that tbb-2 may be specialized for interactions with the cell cortex.

SID-1 Functions in Multiple Roles To Support Parental RNAi in Caenorhabditis elegans.

Systemic RNA interference (RNAi) in Caenorhbaditis elegans requires sid-1, sid-3, and sid-5 Injected, expressed, or ingested double-stranded RNA (dsRNA) is transported between cells, enabling RNAi in most tissues, including the germline and progeny (parental RNAi). A recent report claims that parental RNAi also requires the yolk receptor rme-2 Here, we characterize the role of the sid genes and rme-2 in parental RNAi. We identify multiple independent paths for maternal dsRNA to reach embryos and initiate RNAi. We showed previously that maternal and embryonic sid-1 contribute independently to parental RNAi. Here we demonstrate a role for embryonic sid-5, but not sid-2 or sid-3 in parental RNAi. We also find that maternal rme-2 contributes to but is not required for parental RNAi. We determine that parental RNAi by feeding occurs nearly exclusively in adults. We also introduce 5-ethynyluridine to densely internally label dsRNA, avoiding complications associated with other labeling strategies such as inhibition of normal dsRNA trafficking and separation of label and RNA. Labeling shows that yolk and dsRNA do not colocalize following endocytosis, suggesting independent uptake, and, furthermore, dsRNA appears to rapidly progress through the RAB-7 endocytosis pathway independently of sid-1 activity. Our results support the premise that although sid-1 functions in multiple roles, it alone is central and absolutely required for inheritance of silencing RNAs.