First, Tuschl and colleagues4 tested whether siRNAs could trigger RNAi in mammalian cells, as had been observed in non-mammalian cells. They assayed the ability of siRNA to target various luciferase transgenes, for which gene expression is easily quantified by measuring luminescence. siRNAs were transfected with cationic liposomes into various mammalian tissue culture cells (NIH/3T3, COS-7, HeLa and 293 cells), as well as into a Drosophila cellculture line for comparison. Indeed, the authors observed reproducible, sequencespecific siRNA inhibition in the mammalian cells, with no sign of the nonspecific effects. In contrast, with longer RNAs, luciferase expression was reduced with every dsRNA tested, no matter what its sequence. Superimposed on the nonspecific inhibition was a sequence-specific inhibition, suggesting that both pathways can operate simultaneously. (As shown in Fig. 1, the two pathways probably compete for the long dsRNA.) Importantly, Tuschl and co-workers went on to show that siRNAs are not only effective at targeting the transgene luciferase, but also at targeting naturally occurring, endogenous genes.
Of course, the story is not as neat and tidy as I have described it here. As the authors are careful to point out, inhibition by siRNAs is effective in mammalian cells, but gene expression is not eliminated completely as it is in Drosophila cells. Further, siRNA techniques in mammalian cells have some of the same drawbacks associated with antisense RNA, another technique used to prevent expression of particular genes. In both cases, success depends on the cell type, as well as on the level of expression of the gene to be targeted. That apart, however, RNAi has repeatedly proven itself to be more robust than antisense techniques: it works more often, and typically decreases expression of a gene to lower levels, or eliminates it entirely. And, as Tuschl and colleagues show, even in mammalian cells, siRNAs are effective at concentrations that are several orders of magnitude below the concentrations typically used in antisense experiments.
One of the most important aspects of the new work is the further research it will inspire. Although RNAi works in mouse eggs and embryos11,12, scientists have been reluctant to invest time in applying it to other mammalian cells because of reported problems13,14. Now we will see studies aimed at optimizing the use of siRNAs, as well as at understanding why conventional RNAi, with longer dsRNA, works in eggs and embryos. Might these cells lack the nonspecific pathway?
The RNAi technique has had a huge impact in studies of non-mammalian systems. Use of siRNA in mammalian cells could be just as far-reaching, with the applications extending to functional genomics and therapeutics. But various technical issues must be addressed, especially for large-scale applications. For instance, dsRNA can be delivered to C. elegans by feeding or soaking, but effective delivery of siRNAs to mammalian cells will not be so simple. The analysis of C. elegans phenotypes is aided by short generation times and a wealth of information about the worm’s morphology and behaviour; developing rapid ways to screen mammalian cells, or whole organisms, will take some time and thought.
So far I have discussed RNAi as a technique. But of course the pathway does not exist in cells solely to make life easier for scientists. RNAi is a natural biological pathway, albeit one we don’t quite understand yet. Especially for those with a long-standing interest in the roles of dsRNA, Tuschl and colleagues’ paper is interwoven with information about how RNAi coexists with previously characterized dsRNA pathways. This is especially interesting because dsRNAbinding proteins are usually not sequence specific and will bind any dsRNA. A single dsRNA can interact with proteins of different pathways so that the pathways compete. The different ratios of specific to nonspecific inhibition observed by the authors are probably telling us something about the particular particular constellation of dsRNA-binding proteins in the different cell types and how they compete with RNAi. Regardless of that, the new study shows that one way dsRNA pathways can coexist is to require different lengths of dsRNA. This is good news for cells — and for researchers.
Brenda L. Bass is in the Department of Biochemistry and Howard Hughes Medical Institute, University of Utah, 50 North Medical Drive, Room 211, Salt Lake City, Utah 84132, USA. e-mail: bbass@howard.genetics.utah.edu
1. Fire, A. et al. Nature 391, 806–811 (1998).
2. Barstead, R. Curr. Opin. Chem. Biol. 5, 63–66 (2001).
3. Kim, S. K. Curr. Biol. 11, R85–R87 (2001).
4. Elbashir, S. M. et al. Nature 411, 494–498 (2001).
5. Elbashir, S. M., Lendeckel, W. & Tuschl, T. Genes Dev. 15,188–200 (2001).
6. Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J.Nature 409, 363–366 (2001).
7. Sharp, P. A. Genes Dev. 15, 485–490 (2001).
8. Hunter, T., Hunt, T., Jackson, R. J. & Robertson, H. D. J. Biol.Chem. 250, 409–417 (1975).
9. Manche, L., Green, S. R., Schmedt, C. & Mathews, M. B. Mol.Cell Biol. 12, 5238–5248 (1992).
10.Minks, M. A., West, D. K., Benvin, S. & Baglioni, C. J. Biol.
Chem. 254, 10180–10183 (1979).
11.Wianny, F. & Zernicka-Goetz, M. Nature Cell Biol. 2, 70–75(2000).
12.Svoboda, P., Stein, P., Hayashi, H. & Schultz, R. M. Development127, 4147–4156 (2000).
13.Ui-Tei, K., Zenno, S., Miyata, Y. & Saigo, K. FEBS Lett. 479,79–82 (2000).
14. Caplen, N. J., Fleenor, J., Fire, A. & Morgan, R. A. Gene 252,95–105 (2000).
