RNAi and double-strand RNA

Abstract

Double-strand RNA (dsRNA) is a signal for gene-specific silencing of expression in a number of organisms. This phenomenon was demonstrated recently in Caenorhabditis elegans when dsRNA was injected into the worm and the corresponding gene products disappeared from both the somatic cells of the organism as well as in its F1 progeny (Fire et al. 1998). This RNA interference, RNAi, has been generalized to many genes in C. elegans (Montgomery and Fire 1998; Shi and Mello 1998; Tabara et al. 1998; Timmons and Fire 1998). ds-RNA can also suppress expression of specific genes in plants, a component of the phenomenon called cosuppression (Vionnet et al. 1998; Waterhouse et al. 1998). Two recent reports document dsRNA-mediated interference with expression of specific genes in other organisms. Double-strand RNA produced gene-specific phenotypes in Trypanosoma brucei (Ngo et al. 1998) and, very recently, dsRNA-mediated interference was demonstrated in Drosophila (Kennerdell and Carthew 1998). Thus, the RNAi phenomenon is likely to be a general mechanism for gene regulation and may be critical for many developmental and antiviral processes. Montgomery et al. (1998) have investigated how RNAi suppresses the expression of endogenous genes in C. elegans. dsRNA might conceivably direct mutations of the endogenous genes thus inactivating function. However, the fact that the F2 progeny from RNAi-treated C. elegans generally reverted to normal phenotype argued against nonreversible gene modification. Further sequencing of the targeted locus failed to detect nucleotide differences, direct evidence against a mutational mechanism. Double-strand RNA primarily suppresses gene expression by a post-transcriptional mechanism in C. elegans (Montgomery et al. 1998). A post-transcriptional mechanism was foreshadowed by earlier experiments showing that dsRNAs from sequences in the mature RNA, that is, exons, had RNAi activity, whereas dsRNAs from intron sequences did not (Fire et al. 1998). The most direct evidence for a post-transcriptional effect arises from analysis of RNAi effects on a multiple-gene operon. Such operons are expressed in C. elegans by transcription of long precursor RNAs that are then processed by trans-splicing and cleavage to generate specific mRNAs. The lin-15b and lin-15a genes are part of one operon and both need to be inactivated to generate the multivulva phenotype. Injection of dsRNA from both genes generated the phenotype, whereas injection of dsRNA from either gene alone did not. This strongly indicates that suppression of the upstream gene does not inactivate the downstream gene and thus that the RNAi effect is post-transcriptional. The post-transcriptional effects of RNAi were directly observed using in situ hybridization to follow transcripts of genes suppressed by injection of dsRNA (Montgomery et al. 1998). There was a diminution of nuclear RNA from the suppressed gene as well as a total absence of the specific mRNA in the cytoplasm. This suggests that dsRNA establishes an intracellular state that destroys RNA transcribed and spliced from a specific gene. Both this study and other results are most easily explained if the specific RNA degradation occurs in both the nucleus and the cytoplasm. dsRNA mediated suppression of specific gene expression has also been observed in plants. One demonstration of the phenomenon follows expression in plant cells of a recombinant RNA virus containing exonic sequences of an endogenous cellular gene. Expression of the cellular gene is suppressed in these cells when the recombinant viral RNAs are capable of replicating and not when they are replication incompetent (Angell and Baulcombe 1997). Viral RNA replication involves dsRNA. A similar phenomenon can be observed when a transgene is introduced into plant cells. The endogenous gene corresponding to the transgene can become suppressed (e.g., Vionnet et al. 1998), perhaps due to symmetric transcription of both strands of the transgene. Such symmetric transcription could arise by initiation in flanking sequences due to the presence of fortuitous promoter sites in plasmid DNA. The plant and nematode effects share the property of spreading. Examples of this are striking. Worms fed dsRNA exhibit a strong systemic interference phenotype (Timmons and Fire 1998) and introduction (into plants) of 500-bp fragments of a gene absorbed on the surface of a gold bead projectile can result in suppression of the gene in cells both immediately adjacent to the site penetrated by the bead as well as at very distant sites (Vionnet et al. 1998). The purest demonstration that dsRNA mediates gene silencing in plants is the genetic study of Waterhouse et al. (1998). Transgenic plants were established which expressed either sense or antisense of a gene of the potato 1E-MAIL sharppa@mit.edu; FAX (617) 253-3867.