RNA Interference

A New Powerful Tool for Developmental Biology

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. In this way, one can functionally delete from the embryo any gene that can be cloned.

I. Why Some Experiments Don't Work

When you do an experiment that doesn't work out the way you know it should, the tendency is to blame yourself for sloppy technique or stupidity. Sometimes, though, the result is really nature's way of telling you that there is something going on that nobody had ever thought about before.

That's what happened when experiments designed to enhance a characteristic through molecular techniques actually knocked out that character. Plant biologists seeking to make a more purple petunia added the gene involved in pigment production to an active promoter, placed this construct into petunias, and portions of the petunias turned white (Napoli et al., 1990). Similarly, when biologists seeking to boost the synthesis of orange pigment in fungi introduced extra copies of a gene encoding a carotenoid synthesizing enzyme into Neurospora, the engineered mold became pigmentless. Instead of activating higher syntheses of these pigments, the introduced genes actually supressed pigment production. In the nematode C. elegans, Guo and Kemphues (1995) used an antisense RNA to wipe out the par-1 transcripts from C. elegans embryo. They got the result they hoped to get. However, their control—injecting the sense strand of the RNA into the embryos—produced the same result!

This phenomenon was called co-suppression (by the plant biologists), quelling (by the fungal biologists), and RNA interference (by the animal developmental biologists). Often it is now called post-transcriptional gene silencing (PTGS). It appears that adding either the sense (in extremely large concentrations), antisense, or double-stranded gene into cells can cause the cells to destroy the mRNA homologous to that made by the introduced gene.

This technique allows one to make organisms that are functionally "knockouts" (i.e., null mutants; loss-of-function mutants) for any gene that can be cloned. Whereas knockout technology in mice is laborious and takes a long time, RNAi can be triggered in nematodes by simply soaking the worms in double stranded DNA.

The usefulness of RNAi technology in nematodes is beautifully illustrated on this site.

II. Mechanism for RNAi: How Does it Work?

It does seems like magic, but RNAi is probably the activity of a very ancient anti-viral defense mechanism. In 1998, Fire and Mello discovered that double stranded (dsRNA) shuts down specific protein production even better than antisense or overexpressed sense messages. They hypothesized that double-stranded RNA resembles a stage of viral or transposon replication and that the cell has evolved defense mechanisms to destroy such nucleic acid and any of its products. The initial trigger of such a cellular antiviral immune system would be a double-stranded RNA, an RNA lacking its cap or tail, or conventional RNA produced in exceptionally large quantities.

The host's response is to produce (or activate) RNA endonucleases that would cleave the offending RNA into pieces about 25 nucleotides long (Figure 1). At some stage (probably after cleavage, but not necessarily) this alarm system is amplified by copying this RNA. (An RNA-dependent RNA polymerase has been identified as crucial to this step.) These RNA fragments are now separated into single strands and the antisense strands has the opportunity to bind to target mRNA. These partially double-stranded messages are degraded by a nuclease that appears to be targeted to the mRNA by the 25-nucleotide antisense nucleotide (Hammond et al., 2000). In virally infected cells, this should get rid of both the replicating virus and the viral messages. (So in experiments using dsRNA, it's like giving the cells an allergyocausing an immune attack on something nonpathogenic that gets treated like a danger to the body).

Figure 1
Figure 1   Possible mechanism for post-transcriptional gene silencing. Double stranded RNA is cut into pieces about 25 nucleotides long and the pieces are amplified. (Alternatively, the amplification might precede the cleavage). These amplified double-stranded oligonucleotides are made single-stranded. The antisense strand can bind to the complementary site of a natural mRNA, targeting it for degradation by a specific RNA nuclease. (After Gura, 2000.)

III. Morpholino Oligigonucleotides

Another new modification of antisense technology is the morpholino oligo (Summerton and Weller, 1997). These differ from traditional antisense oligonucleotides in that they contain six-member morpholine rings instead of five-member ribose or deoxyribose sugars. This gives them several advantages over their DNA and RNA counterparts. First, they have complete resistance to nucleases. Therefore, they stay in the cell and function longer. They also can hybridize with their target mRNAs independently of the salt concentration and over a large concentration range. Moreover, they do not bind to albumin like DNA and RNA oligos, so they can be used effectively in the presence of serum (in cell culture). The stability of these morpholino antisense oligos allows them to be injected into a cell and then effect events many cell generations afterward (Heasman et al., 2000).

Literature Cited

Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 706 - 811.

Guo, S., and Kemphues, K. 1995 par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative ser/thr kinase that is asymmetrically distributed. Cell 81: 611-620.

Gura, T. 2000. A silence that speaks volumes. Nature 404: 804 - 808.

Hammond, S. M., Bernstein, E., Beach, D., and Hannon, G. J. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404: 293 - 296.

Heasman, J., Kofron, M.,and Wylie, C. 2000. beta-catenin signaling activity dissected in the early Xenopus embryo: A novel antisense approach. Dev. Biol. 222: 124-134.

Napoli, C., Lemieux, C., and Jorgensen, R. A. 1990. Introduction of a chimeric chalcone gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279 - 289.

Summerton, J. and Weller, D. 1997. Morpholino antisense oligomers: design, preparation, and properties. Antisense and Nucleic Acid Drug Development 7:187-95.

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