682 research outputs found
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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.Molecular and Cellular Biolog
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Two Classes of Silencing RNAs Move between Caenorhabditis elegans Tissues
Organism-wide RNA interference (RNAi) is due to the transport of mobile silencing RNA throughout the organism, but the identities of these mobile RNA species in animals are unknown. Here, we present genetic evidence that both the initial double-stranded RNA (dsRNA), which triggers RNAi, and at least one dsRNA intermediate produced during RNAi can act as or generate mobile silencing RNA in C. elegans. This dsRNA intermediate requires the long dsRNA-binding protein RDE-4, the endonuclease DCR-1, which cleaves long dsRNA into double-stranded short-interfering RNA (ds-siRNA), and the putative nucleotidyltransferase MUT-2 (RDE-3). However, single-stranded siRNA and downstream secondary siRNA produced upon amplification by the RNA-dependent RNA polymerase RRF-1 do not generate mobile silencing RNA. Restricting intertissue transport to long dsRNA and directly processed siRNA intermediates rather than amplified siRNA may serve to modulate the extent of systemic silencing in proportion to available dsRNA.Molecular and Cellular Biolog
Synthetic lethal analysis of Caenorhabditis elegans posterior embryonic patterning genes identifies conserved genetic interactions
Phenotypic robustness is evidenced when single-gene mutations do not result in an obvious phenotype. It has been suggested that such phenotypic stability results from 'buffering' activities of homologous genes as well as non-homologous genes acting in parallel pathways. One approach to characterizing mechanisms of phenotypic robustness is to identify genetic interactions, specifically, double mutants where buffering is compromised. To identify interactions among genes implicated in posterior patterning of the Caenorhabditis elegans embryo, we measured synthetic lethality following RNA interference of 22 genes in 15 mutant strains. A pair of homologous T-box transcription factors (tbx-8 and tbx-9) is found to interact in both C. elegans and C. briggsae, indicating that their compensatory function is conserved. Furthermore, a muscle module is defined by transitive interactions between the MyoD homolog hlh-1, another basic helix-loop-helix transcription factor, hnd-1, and the MADS-box transcription factor unc-120. Genetic interactions within a homologous set of genes involved in vertebrate myogenesis indicate broad conservation of the muscle module and suggest that other genetic modules identified in C. elegans will be conserved
Composition and regulation of maternal and zygotic transcriptomes reflects species-specific reproductive mode
Background
Early embryos contain mRNA transcripts expressed from two distinct origins; those expressed from the mother's genome and deposited in the oocyte (maternal) and those expressed from the embryo's genome after fertilization (zygotic). The transition from maternal to zygotic control occurs at different times in different animals according to the extent and form of maternal contributions, which likely reflect evolutionary and ecological forces. Maternally deposited transcripts rely on post-transcriptional regulatory mechanisms for precise spatial and temporal expression in the embryo, whereas zygotic transcripts can use both transcriptional and post-transcriptional regulatory mechanisms. The differences in maternal contributions between animals may be associated with gene regulatory changes detectable by the size and complexity of the associated regulatory regions.
Results
We have used genomic data to identify and compare maternal and/or zygotic expressed genes from six different animals and find evidence for selection acting to shape gene regulatory architecture in thousands of genes. We find that mammalian maternal genes are enriched for complex regulatory regions, suggesting an increase in expression specificity, while egg-laying animals are enriched for maternal genes that lack transcriptional specificity.
Conclusions
We propose that this lack of specificity for maternal expression in egg-laying animals indicates that a large fraction of maternal genes are expressed non-functionally, providing only supplemental nutritional content to the developing embryo. These results provide clear predictive criteria for analysis of additional genomes.Molecular and Cellular Biolog
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Early Developmental Exposure to dsRNA Is Critical for Initiating Efficient Nuclear RNAi in C.Ā elegans
RNAi has enabled researchers to study the function of many genes. However, it is not understood why some RNAi experiments succeed while others do not. Here, we show in C. elegans that pharyngeal muscle is resistant to RNAi when initially exposed to double-stranded RNA (dsRNA) by feeding but sensitive to RNAi in the next generation. Investigating this observation, we find that pharyngeal muscle cells as well as vulval muscle cells require nuclear rather than cytoplasmic RNAi. Further, we find in these cell types that nuclear RNAi silencing is most efficiently triggered during early development, defining a critical period for initiating nuclear RNAi. Finally, using heat-shock-induced dsRNA expression, we show that synMuv B class mutants act in part to extend this critical window. The synMuv-B-dependent early-development-associated critical period for initiating nuclear RNAi suggests that mechanisms that restrict developmental plasticity may also restrict the initiation of nuclear RNAi.Molecular and Cellular Biolog
The homeodomain protein PAL-1 specifies a lineage-specific regulatory network in the C. elegans embryo
Maternal and zygotic activities of the homeodomain protein PAL-1 specify the identity and maintain the development of the multipotent C blastomere lineage in the C. elegans embryo. To identify PAL-1 regulatory target genes, we used microarrays to compare transcript abundance in wild-type embryos with mutant embryos lacking a C blastomere and to mutant embryos with extra C blastomeres. pal-1-dependent C-lineage expression was verified for select candidate target genes by reporter gene analysis, though many of the target genes are expressed in additional lineages as well. The set of validated target genes includes 12 transcription factors, an uncharacterized wingless ligand and five uncharacterized genes. Phenotypic analysis demonstrates that the identified PAL-1 target genes affect specification, differentiation and morphogenesis of C-lineage cells. In particular, we show that cell fate-specific genes (or tissue identity genes) and a posterior HOX gene are activated in lineage-specific fashion. Transcription of targets is initiated in four temporal phases, which together with their spatial expression patterns leads to a model of the regulatory network specified by PAL-1
MEX-3 interacting proteins link cell polarity to asymmetric gene expression in Caenorhabditis elegans
The KH domain protein MEX-3 is central to the temporal and spatial control of PAL-1 expression in the C. elegans early embryo. PAL-1 is a Caudal-like homeodomain protein that is required to specify the fate of posterior blastomeres. While pal-1 mRNA is present throughout the oocyte and early embryo, PAL-1 protein is expressed only in posterior blastomeres, starting at the four-cell stage. To better understand how PAL-1 expression is regulated temporally and spatially, we have identified MEX-3 interacting proteins (MIPs) and characterized in detail two that are required for the patterning of PAL-1 expression. RNA interference of MEX-6, a CCCH zinc-finger protein, or SPN-4, an RNA recognition motif protein, causes PAL-1 to be expressed in all four blastomeres starting at the four-cell stage. Genetic analysis of the interactions between these mip genes and the par genes, which provide polarity information in the early embryo, defines convergent genetic pathways that regulate MEX-3 stability and activity to control the spatial pattern of PAL-1 expression. These experiments suggest that par-1 and par-4 affect distinct processes. par-1 is required for many aspects of embryonic polarity, including the restriction of MEX-3 and MEX-6 activity to the anterior blastomeres. We find that PAL-1 is not expressed in par-1 mutants, because MEX-3 and MEX-6 remain active in the posterior blastomeres. The role of par-4 is less well understood. Our analysis suggests that par-4 is required to inactivate MEX-3 at the four-cell stage. Thus, PAL-1 is not expressed in par-4 mutants because MEX-3 remains active in all blastomeres. We propose that MEX-6 and SPN-4 act with MEX-3 to translate the temporal and spatial information provided by the early acting par genes into the asymmetric expression of the cell fate determinant PAL-1
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Synthetic Lethal Analysis of Caenorhabditis elegans Posterior Embryonic Patterning Genes Identifies Conserved Genetic Interactions
Phenotypic robustness is evidenced when single-gene mutations do not result in an obvious phenotype. It has been suggested that such phenotypic stability results from 'buffering' activities of homologous genes as well as non-homologous genes acting in parallel pathways. One approach to characterizing mechanisms of phenotypic robustness is to identify genetic interactions, specifically, double mutants where buffering is compromised. To identify interactions among genes implicated in posterior patterning of the Caenorhabditis elegans embryo, we measured synthetic lethality following RNA interference of 22 genes in 15 mutant strains. A pair of homologous T-box transcription factors (tbx-8 and tbx-9) is found to interact in both C. elegans and C. briggsae, indicating that their compensatory function is conserved. Furthermore, a muscle module is defined by transitive interactions between the MyoD homolog hlh-1, another basic helix-loop-helix transcription factor, hnd-1, and the MADS-box transcription factor unc-120. Genetic interactions within a homologous set of genes involved in vertebrate myogenesis indicate broad conservation of the muscle module and suggest that other genetic modules identified in C. elegans will be conserved.Molecular and Cellular Biolog
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Evaluation of Normalization Procedures for Oligonucleotide Array Data Based On Spiked cRNA Controls
Background: Affymetrix oligonucleotide arrays simultaneously measure the abundances of thousands of mRNAs in biological samples. Comparability of array results is necessary for the creation of large-scale gene expression databases. The standard strategy for normalizing oligonucleotide array readouts has practical drawbacks. We describe alternative normalization procedures for oligonucleotide arrays based on a common pool of known biotin-labeled cRNAs spiked into each hybridization. Results: We first explore the conditions for validity of the 'constant mean assumption', the key assumption underlying current normalization methods. We introduce 'frequency normalization', a 'spike-in'-based normalization method which estimates array sensitivity, reduces background noise and allows comparison between array designs. This approach does not rely on the constant mean assumption and so can be effective in conditions where standard procedures fail. We also define 'scaled frequency', a hybrid normalization method relying on both spiked transcripts and the constant mean assumption while maintaining all other advantages of frequency normalization. We compare these two procedures to a standard global normalization method using experimental data. We also use simulated data to estimate accuracy and investigate the effects of noise. We find that scaled frequency is as reproducible and accurate as global normalization while offering several practical advantages. Conclusions: Scaled frequency quantitation is a convenient, reproducible technique that performs as well as global normalization on serial experiments with the same array design, while offering several additional features. Specifically, the scaled-frequency method enables the comparison of expression measurements across different array designs, yields estimates of absolute message abundance in cRNA and determines the sensitivity of individual arrays.Molecular and Cellular Biolog
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