RNAi in other systems
Mechanism of RNAi
Current RNAi studies
RNAi protocols from the FireLab at the Carnegie Institution of Washington
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:
dsRNA, rather than single-stranded antisense RNA, is the interfering agent
it is highly specific
it is remarkably potent (only a few dsRNA molecules per cell are required for effective interference)
the interfering activity (and presumably the dsRNA) can cause interference in cells and tissues far removed from the site of introduction
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. ( Embryo from uninjected parent showing normal pattern of endogenous mex-3 RNA (purple staining). © 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 Ám 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
Drosophila (Kennerdell, JR and RW Carthew '98 Development 95: 1017-26; Misquitta, L and BM Paterson '99 PNAS 96: 1451-56)
trypanosomes (Ngo, H et al. '98 PNAS 95: 14687-92)
planaria (Newmark, P and A. Sanchez '99 PNAS 96: 5049-54 )
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
The mechanism of how dsRNA results in the loss of the targeted homologous mRNA is still not well understood. A number of observations indicate 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
Because RNAi is also remarkably potent (i.e., only a few dsRNA molecules per cell are required to produce effective interference), the dsRNA must be either replicated and/or work catalytically. The current model favors a catalytic mechanism by which the dsRNA unwinds slightly, allowing the antisense strand to base pair with a short region of the target endogenous message and marking it for destruction. "Marking" mechanisms could involve covalent modification of the target (e.g. by adenosine deaminase) or any number of other mechanisms. Potentially, a single dsRNA molecule could mark hundreds of target mRNAs for destruction before it itself is "spent."
Current RNAi Studies
USING RNAi TO STUDY MATERNAL GENE FUNCTION IN "NON-MODEL" NEMATODES
We are interested in comparing the role of maternal factors in early pattern formation between C. elegans and related nematode species, including the closely related C. briggsae and more distantly related Cephalobus sp. We are especially interested in maternal genes involved in cell-cell interactions affecting cell fate, such as apx-1, which encodes the ligand for GLP-1 (a Notch homolog), and mediates signalling from the germline blastomere P2 to a somatic blastomere, ABp, in C. elegans. Whereas the division patterns and blastomere positions in the early C. briggsae embryo closely resemble those of C. elegans (e.g., P2 still contacts ABp in a similar manner), those inCephalobus are significantly different. Thus, we would like to know to what extent the mechanisms governing cell fate in the early embryo have been conserved (or altered) in nematode development. One approach we are using is to clone homologs of C. elegans maternal genes, such as those in the Notch signalling pathway, from C. briggsae and Cephalobus, and use the powerful tool of RNAi to disrupt gene function of the homologs in these genetically less tractable species.
Although first demonstrated in C. elegans, because RNAi has proven effective in such phylogenetically diverse species as Drosophila, trypanosomes, and planaria, we believe RNAi might be useful to study gene function in many "non-model" organisms as well.
We are also working on determining what percentage of sequence identity is needed between the exogenous dsRNA and the target endogenous message to achieve effective interference.
Figure 2. SOAKING WORMS WORKS ALMOST AS EFFECTIVELY AS INJECTING. These images are from an experiment performed by Jeff Norman (currently a student in the lab), 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.
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