Double-stranded RNA-mediated gene interference (RNAi) in Caenorhabditis elegans systemically inhibits gene expression throughout the organism. To investigate how gene-specific silencing information is transmitted between cells, we constructed a strain that permits visualization of systemic RNAi. We used this strain to identify systemic RNA interference-deficient (sid) loci required to spread gene-silencing information between tissues but not to initiate or maintain an RNAi response. One of these loci, sid-1, encodes a conserved protein with predicted transmembrane domains. SID-1 is expressed in cells sensitive to RNAi, is localized to the cell periphery, and is required cell-autonomously for systemic RNAi.

RNA interference (RNAi) spreads systemically in plants and nematodes to silence gene expression distant from the site of initiation. We previously identified a gene, sid-1, essential for systemic but not cell-autonomous RNAi in Caenorhabditis elegans. Here, we demonstrate that SID-1 is a multispan transmembrane protein that sensitizes Drosophila cells to soaking RNAi with a potency that is dependent on double-stranded RNA (dsRNA) length. Further analyses revealed that SID-1 enables passive cellular uptake of dsRNA. These data indicate that systemic RNAi in C. elegans involves SID-1-mediated intercellular transport of dsRNA.

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

In plants and in the nematode Caenorhabditis elegans, an RNAi signal can trigger gene silencing in cells distant from the site where silencing is initiated. In plants, this signal is known to be a form of dsRNA, and the signal is most likely a form of dsRNA in C. elegans as well. Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). Ingestion and soaking delivery of dsRNA has also been described for other invertebrates. Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. SID-2, when expressed in the environmental RNAi defective species Caenorhabditis briggsae, confers environmental RNAi.dsRNA ͉ transmembrane ͉ intestine lumen T ranslocation of nucleic acids across cellular membranes is associated with viral infection, bacterial conjugation, and transport of nuclear encoded tRNAs into mitochondria (1-3). In these cases, specific machinery acts to translocate a specific RNA or DNA across one or more membranous barriers. However, observations in plants suggest the routine intercellular transport of cellular mRNAs as well as processed, likely short, dsRNAs associated with RNAi-related phenomena that mediate systemic virus resistance (4, 5). In the nematode Caenorhabditis elegans, RNA-induced gene silencing is also systemic, spreading from the site of injection or expression to silence the targeted gene throughout the animal and in its progeny (6, 7). Furthermore, RNAi can be initiated by soaking animals in solutions of dsRNA or feeding worms bacteria expressing dsRNA (8, 9). Thus dsRNA can enter the animal from the environment. RNAi triggered by environmental exposure to dsRNA has also been documented in planaria, moth, tick, hydra, and numerous parasitic nematodes (10-15), suggesting that many invertebrates possess mechanisms to transport sequence nonspecific dsRNA into and between cells.To identify cellular components required for systemic RNAi in C. elegans, we isolated systemic RNAi defective (Sid) mutants defective for spreading of RNAi (7). The first characterized gene, sid-1, encodes a transmembrane protein expressed in all cells sensitive to systemic RNAi and is required for uptake of dsRNA (7, 16). Furthermore, SID-1 expressed in Drosophila S2 cells is sufficient to mediate passive uptake of dsRNA from the growth media, indicating that SID-1 most likely acts as a channel for diffusion of dsRNA into cells (16). These observations provide strong support for the notion that dsRNA is systemically transported in C. elegans.A SID-1::GFP fusion reporter construct that rescues sid-1 mutant worms was expressed at highest levels in cells directly exposed to the environment (7). This observation suggested that environmental dsRNA might enter the animal via these sid-1 expressing cells. Here we describe sid-2, a gene specifically required for uptake of silencing information (hereafter assumed to be dsRNA) from the environment...

Temporal profiles of transcript abundance during embryonic development were obtained by whole-genome expression analysis from precisely staged C. elegans embryos. The result is a highly resolved time course that commences with the zygote and extends into mid-gastrulation, spanning the transition from maternal to embryonic control of development and including the presumptive specification of most major cell fates. Transcripts for nearly half (8890) of the predicted open reading frames are detected and expression levels for the majority of them (>70%) change over time. The transcriptome is stable up to the four-cell stage where it begins rapidly changing until the rate of change plateaus before gastrulation. At gastrulation temporal patterns of maternal degradation and embryonic expression intersect indicating a mid-blastula transition from maternal to embryonic control of development. In addition, we find that embryonic genes tend to be expressed transiently on a time scale consistent with developmental decisions being made with each cell cycle. Furthermore, overall rates of synthesis and degradation are matched such that the transcriptome maintains a steady-state frequency distribution. Finally, a versatile analytical platform based on cluster analysis and developmental classification of genes is provided.