Transport of sequence-specific RNA interference information between cells

Abstract

When eukaryotic cells encounter double-stranded RNA, genes of matching sequence are silenced through RNA interference. Surprisingly, in some animals and plants, the same gene is specifically silenced even in cells that did not encounter the double-stranded RNA, due to the transport of a gene-specific silencing signal between cells. This silencing signal likely has an RNA component that gives it sequence-specificity, however its precise identity remains unknown. Studies in the worm Caenorhabditis elegans and in plants have revealed parts of a complex protein machinery that transports this silencing signal. Some of these proteins are conserved in vertebrates, including mammals, raising the possibility that higher animals can communicate gene-specific silencing information between cells. Such communication provides antiviral immunity in plants and perhaps in C. elegans. Identifying the transported silencing signal and deciphering the evolutionarily selected role of the transport machinery are some of the key challenges for the future.

Figure 1

The transport of silencing information between cells can be triggered in many ways in C. elegans. (a) When worms are either fed bacteria that express dsRNA or soaked in a dsRNA solution, the dsRNA is imported from the gut lumen into gut cells, and is disseminated to distant sites such as body-wall muscles and the reproductive system, presumably through the pseudocoelomic fluid (not shown), which bathes all tissues. Transcription start sites (small black arrows) and DNA (bacterial plasmid, PCR product, or inverted-repeat transgene) with target gene are shown. (b) Hairpin dsRNA made in vivo in the pharynx and (c) dsRNA injected into single gut cells likely also cause silencing to spread to other tissues via the pseudocoelom.

Figure 2

Part of the molecular machinery that controls the uptake of dsRNA and mediates intercellular transport of silencing in C. elegans is known. A cross-section through the worm shows the transport of dsRNA and a dsRNA-derived silencing signal during RNAi. dsRNA is first imported from the gut lumen into gut cells. Imported dsRNA is either converted into a derived silencing signal and then exported or is directly exported out of gut cells into the pseudocoelom. The silencing signal is finally imported into the gonad and into body-wall muscles and other somatic tissues. The flowchart on the right shows the steps and proteins required during the intercellular transport of silencing. SID-1/RSD-8 is a dsRNA channel; SID-2/FED-1/RSD-4 is a transmembrane protein located in the lumenal membrane of the intestine; RSD-2 and RSD-6 form a protein complex that may bind RNA; and RSD-3 may mediate endocytosis. The silencing signal derived from in vivo expression of dsRNA or injection of dsRNA into a single gut cell is also presumably exported into the pseudocoelom from which it is imported into cells as above (not shown).

Figure 3

Plants use distinct mechanisms for cell-to-cell and long-distance transport of silencing information. (a) Schematic showing the organization of plant vasculature. Long-distance transport of silencing information occurs through the phloem, comprised of companion cells that support associated sieve elements. Cell-to-cell transport in leaves occurs through plasmodesmal connections, which connect the cytoplasm of all cells except guard cells that surround sites of gaseous exchange (stomata). (b) Model for the generation and amplification of a cell-to-cell silencing signal. The RNAi trigger, long dsRNA, is converted to 21-nt siRNAs by DCL4, and modification of these 21-nt siRNAs for transport and/or their transport through plasmodesmata is controlled by the silencing movement defective genes (SMD1, 2, 3). This signal can travel for ~10–15 cells without any amplification. However, in the presence of target mRNA, a cellular RNA-dependent RNA polymerase (RDR6) and an RNA helicase (SDE3) can direct the synthesis of secondary dsRNA, which may then be processed just as the trigger long dsRNA. (c) Model for the long-distance transport of a silencing signal. The silencing signal (likely RNA) may be transported as single-stranded short RNAs through the phloem since the phloem small RNA-binding protein 1 (PSRP1) of pumpkin specifically binds short single-stranded RNAs. Similar PSRPs may exist in other plants.