RNAi

Background
RNAi in other systems
Mechanism of RNAi
Current RNAi studies
RNAi protocols from the FireLab at the Carnegie Institution of Washington


Background

RNAi (RNA interference) refers to the introduction of homologous double stranded RNA (dsRNA) to specifically target a gene's product, resulting in null or hypomorphic phenotypes. The use of antisense RNA to interfere with a gene's activity in C. elegans was first utilised by Su Guo and Ken Kemphues to study par-1 ; however, it was reported that control sense RNA also produced a par-1 mutant phenotype (Cell 81: 611-20, 1995). Subsequently, it was discovered by Fire et al. '98 that it is the presence of dsRNA, formed from the annealing of sense and antisense strands present in the in vitro RNA preps, that is responsible for producing the interfering activity. Introduction of dsRNA into an adult worm results in the loss of the targeted endogenous mRNA from both the adult and its progeny. This phenomenon has been effectively harnessed to study an ever increasing number of maternal and zygotic genes in C. elegans.

The most interesting aspects of RNAi are the following:

Figure 1. Effects of mex-3 RNA interference on levels of the endogenous mRNA. Nomarski DIC micrographs show in situ hybridization of 4-cell stage embryos. (A) Negative control showing lack of staining in the absence of the hybridization probe. (B) Embryo from uninjected parent showing normal pattern of endogenous mex-3 RNA (purple staining). (C) Embryo from parent injected with purified mex-3 antisense RNA. These embryos (and the parent animals) retain mex-3 mRNA, although levels may be somewhat less than wild type. (D) Late 4-cell stage embryo from a parent injected with dsRNA corresponding to mex-3 ; no mex-3 RNA is detected. (Templates used for interfering RNA and in situ probes were largely non-overlapping.)
Each embryo is approximately 50 µm in length.
(For details see: Fire et al. '98 "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans " Nature 391: 806-11)

RNAi in Other Systems

More surprisingly, introduction of dsRNA has been recently shown to produce specific phenocopies of null mutations in such phylogenetically diverse organisms as

The phenomenon of post-transcriptional gene silencing observed in PLANTS may also be due to a related RNAi mechanism. See Waterhouse et al. '98 "Virus resistance and gene silencing in plants can be induced by simulataneous expression of sense and antisense RNA" PNAS 95: 13959-64.

(Also, for recent review of the field, see Sharp, P '99 Genes & Development 13: 139-41)

Mechanism of RNAi

In recent years several studies have shed light on the underlying mechanisms of how dsRNA results in the loss of the targeted homologous mRNA. Early observations indicated that the primary interference effects are post-transcriptional. First it was observed by Craig Mello, and reported in Fire et al. ('98), that only dsRNA targeting exon sequences was effective (promoter and intron sequences could not produce an RNAi effect). Additional evidence supporting mature messages as the most likely target of RNA-mediated interference is summarised below (from Montgomery et al. ‘98, PNAS 95: 15502-07):
 
*       primary DNA sequence of target appears unaltered
*       initiation and elongation of transcription appear unaffected
*       nascent transcripts can be detected but are apparently degraded before leaving the nucleus
 
RNAi is remarkably potent (i.e., only a few dsRNA molecules per cell are required to produce effective interference).  This observation suggested that the dsRNA must be either replicated and/or function catalytically; models that have been mutually supported by further studies. Genetic and biochemical studies involving plants and flies as well as worms have uncovered similar processes in which the dsRNA is cleaved into ~23 bp short interfering RNAs (siRNAs) by an enzyme called Dicer (Bernstein et al., 2001; Hamilton & Baulcombe, 1999, Science 286: 950), thus producing multiple “trigger” molecules from the original single dsRNA.  The siRNA-Dicer complex recruits additional components to form an RNA-induced Silencing Complex (RISC) in which the unwound siRNA base pairs with complementary mRNA, thus guiding the RNAi machinery to the target mRNA resulting in the effective cleavage and subsequent degradation of the mRNA (Hammond et al., 2000, Nature 404: 293-96, Zamore et al., 2000, Cell 101: 25-33; Pham et al., 2004, Cell 117: 83-94).  In this way, the activated RISC could potentially target multiple mRNAs, and thus function catalytically. 
 
In addition, a role for RNA-dependent RNA polymerases (RdRP) has been found for some species; mutations have been shown to effect the RNAi response as well as result in increased viral susceptibility, and/or developmental defects (reviewed in Hutvagner & Zamore, 2002, Curr. Opin. Genet. Dev. 12: 225-32).  This ability to generate dsRNA de novo supports the replication hypothesis.


Current RNAi Studies

DIFFERING SUSCEPTIBILITIES TO SYSTEMIC RNA INTERFERENCE WITHIN SOIL NEMATODES OF THE GENUS CAENORHABDITIS

Veronic Descotte and Mary K. Montgomery

Although certain features of the mechanism responsible for the RNAi response appear evolutionarily conserved (e.g. Dicer), the phenomenon of "spreading" exhibited by C. elegans, whereby the worm produces a systemic response to the localized introduction of dsRNA, is more species-specific. Delivery of dsRNA into C. elegans by microinjection (Fire et al., Nature 391: 806), soaking (Tabara et al., Science 282: 430), or feeding (Timmons & Fire, Nature 395: 854) can lead to the systemic depletion of targeted mRNAs, with the exception of a few resistant cell types (e.g. most neurons). The presence of this systemic response lead to the hypothesis that an uptake mechanism must exist that functions to transport the dsRNA or a related RNA product from one cell or tissue to another (e.g. from the gut to the germ line and other tissues). Although a similar spreading phenomenon has been documented in plants, systemic RNAi does not appear to be a common feature in other animals, such as Drosophila. In experiments designed to identify conditions that would result in "cross-interference" (the targeting of a mRNA by a dsRNA containing sequence mismatches), we noticed that the strain of C. briggsae with which we were working was RNAi-resistant via soaking but not by injection. This lead to our testing of a dozen strains within the Caenorhabditis clade for susceptibility to RNAi by different delivery methods (injection, soaking, and feeding). Our results indicate that most strains are capable of a robust systemic RNAi response if dsRNA is delivered via microinjection. However, attempts to target maternal or embryonic mRNAs via soaking or feeding of L4s and adults consistently failed in the seven C. briggsae, three C. remanei, and two C. sp. strains tested. Thus, these results suggest that a separate or additional uptake mechanism may be required to transport the initial dsRNA across epithelial boundaries into the cytoplasm, and that further transport to other cells/tissues represents a second transport mechanism. Some strains may be deficient in the first mechanism but not the second. Our results also suggest that the many nematode species outside the Caenorhabditis clade that have been labeled "RNAi-resistant" may, like the majority of C. elegans neurons, simply lack the transport machinery needed for a systemic RNAi response, but may still be capable of cell autonomous RNAi.

 

Figure 2. SOAKING C. elegans WORKS ALMOST AS EFFECTIVELY AS INJECTING. These images are from an experiment performed by Jeff Norman (in 1999), demonstrating the results of mex-3 in situ hybridization following an RNAi soaking protocol (for original methods, see Tabara et al. '98 Science 282: 430-31; specific protocol used in this experiment was obtained from K. Subrumaniam in Geraldine Seydoux's lab). The left panels show the wildtype pattern of endogenous mex-3 mRNA in untreated adults and embryos. The right panels show loss of mex-3 staining following soaking of L4 hermaphrodites overnight in mex-3 dsRNA. Endogenous mex-3 RNA is greatly reduced, although still faintly detectable; this experiment resulted in approximately 90% dead embryos. Although not as effective as directly injecting dsRNA, this approach is VASTLY EASIER and may be good enough for analysis of most maternally acting genes. Soaking as a delivery method, however, does NOT work for many other nematode species.

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