RNA interference and the mechanism of messenger RNA degradation
By Mehdi Banan, Ambion, Inc.
Table of Contents
The history…
Mechanism of action: the evidence
Mechanism of action: the models
The lingering questions
Post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) has captured the imagination of scientists working on organisms ranging from Drosophila to Neurospora to petunias. While the natural gene silencing functions of RNAi are fascinating, potential uses of RNAi are perhaps even more exciting. By injecting dsRNA into tissues one could possibly inactivate specific genes in specific tissues and, by timing the injections appropriately, could even target a particular stage of development.
The history…
A puzzling phenomenon has added another entry to the list of eukaryotic modes of gene regulation. This occurrence, known as post-transcriptional gene silencing (PTGS) or RNA interference (RNAi), has captured the imagination of scientists working on organisms ranging from Drosophila to Neurospora to petunias. The basic premise of RNAi is the ability of double-stranded RNA (dsRNA) to specifically block expression of its homologous gene when injected into cells. The discovery of this incidence came with the observation that injection of antisense or sense RNA strands into Caenorhabditis elegans cells could result in gene-specific inactivation (1). While gene inactivation by the antisense strand was expected, gene silencing by the sense strand certainly came as a surprise. Adding to the surprise was the finding that this gene-specific inactivation actually came from trace amounts of contaminating dsRNA (2).
Since then, this mode of post-transcriptional gene silencing has been tied to a wide variety of organisms: plants, flies, trypanosomes, planaria, hydra, zebrafish, and mice (3,4). The innate functions of RNAi could be just as varied as the organisms involved. These double stranded RNA molecules have been associated with functions as disparate as transposon-silencing, anti-viral defense mechanisms, and gene regulation (5).
While the natural functions of RNAi are fascinating, its potential uses are just as exciting. By injecting dsRNA into tissues, one could possibly inactivate specific genes not only in those tissues, but also during various stages of development. This is in contrast to tissue-specific knockouts or tissue-specific dominant-negative gene expressions, which do not allow for gene silencing during various stages of the developmental process (4).
Mechanism of action: the evidence
As exciting as these research possibilities are, the uncertainty of the mechanism of RNAi action has deterred some scientists from its use. Recent experiments by Hammond et al. (6) and Zamore et al. (3), however, should alleviate some of these concerns. These groups have given new insights into how dsRNA leads to degradation of its homologous mRNA: namely that the double-stranded RNA is cut by a nuclease activity into 21-23 nucleotide fragments. These fragments, in turn, target the homologous region of their corresponding mRNA, hybridize, and result in a double-stranded substrate for a nuclease that degrades it into fragments of the same size.
Hammond et al. used a cell-free assay system whereby Drosophila embryonic cells were transiently transfected with a given dsRNA. These lysates were capable of degrading synthetic mRNAs homologous to the injected dsRNA. Treatment of the lysates with micrococcal nuclease (which degrades DNA and RNA) but not DNAse I (which degrades DNA) abolished the mRNA-specific nuclease activity, suggesting that RNA was essential for the degradation process. Moreover, it was shown that proteins were also necessary for this mRNA-specific nuclease activity.
The mRNA-specific nuclease activity was purified from lysates using anion-exchange chromatography. Northern blots showed that the active fractions contained an RNA species of approximately 25 nucleotides, homologous to that of the injected double stranded RNA.
These experiments suggested that the dsRNA was first degraded into smaller pieces by a nuclease. These small RNA fragments, along with a nuclease (possibly the one responsible for degradation of the dsRNA), subsequently targeted the cognate mRNA for degradation.
The experiments by Zamore et al. gave added details of the RNA interference process. In contrast to Hammond et al., these scientists used a cell-free system whereby lysates were prepared from Drosophila embryos lacking prior dsRNA injections. These lysates were capable of processing dsRNA into 21-23 nucleotide fragments by cutting them at either the sense or the antisense strand.
An interesting additional insight came when the lysates were incubated with a labeled mRNA and various dsRNAs spanning the message. It was revealed that the dsRNAs determined the boundary of digestion. Moreover, the mRNA was also digested into 21-23 nucleotides fragments with a preference for uracils, suggesting that the small 21-23 nucleotide dsRNAs were tagging the complementary mRNA sites for cleavage.
Mechanism of action: the models
These experiments have led to two proposed models for RNAi activity (see figure). In the "dissociative model", digested 21-23 nucleotide dsRNAs are first dissociated into ssRNAs. These ssRNAs in turn target the homologous region of the cognate mRNA for digestion (3). In the "associative model", the complementary region of the mRNA displaces the sense strand of the 21-23-nucleotide dsRNA, whereby the mRNA is subsequently targeted and digested (7).
The above models suggest a need for a nuclease activity (for cleaving the dsRNA and mRNA) and a helicase activity (for dissociating the dsRNA). Although these genes have yet to be identified, some candidates are in place. The C. elegans mut-1 gene, which has been implicated in RNAi function, has homology to ribonuclease D (8). The Neurospora qde-3 gene, also implicated in RNAi activity, has homology to a DNA-helicase gene family (9).
The lingering questions
These experiments provide very interesting insights into the mechanism of RNA interference. While we now have a better understanding of the biochemistry of RNA interference, the picture is far from complete. Which of the two models (associative or dissociative) is correct? What are the enzymes involved in the various stages of this process?
In addition, the relationship of these small dsRNA fragments to the biological functions of RNAi is unclear. How do these small RNA pieces, for example, prevent transposon hopping? How abundant are these RNA pieces and how are they generated? Yet what does remain clear is that RNA interference is a very interesting research field that will bring excitement to the scientific community for years to come.
References
- Guo, S., and Kempheus, K.J. (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.
- 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, 806-811.
- Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D. P. (2000). RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33.
- Gura, T. (2000). A silence that speaks volumes. Nature 404, 804-808.
- Grant, S. (1999). Dissecting the mechanisms of posttranscriptional gene silencing: divide and conquer. Cell 96, 303-306.
- Hammond, S., Bernstein, E., Beach, D., and Hannon, G. (2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 404, 293-298.
- Bass, B.L. (2000). Double-stranded RNA as a template for gene silencing. Cell 101, 235-238.
- Ketting, R.F., Haverkamp, T.H., van Luenen, H.G., and Plasterk, R.H. (1999). Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNase D. Cell 99, 133-141.
- Cogoni, C., and Macino, G. (1999). Posttranscriptional gene silencing in Neurospora crassa by a RecQ DNA helicase. Science 286, 2342-2344.
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