|
|
楼主 |
发表于 2004-11-26 18:39:29
|
显示全部楼层
RNAi - a review
Discovery of RNA Interference (RNAi)
Recently scientists working in different research fields observed a phenomenon they could not immediately understand. Plant biologists were attempting to boost the activity of the gene for chalcone synthase, an enzyme involved in the production of anthocyanin pigments, by introducing a powerful promotor sequence into their petunias. However, instead of a deep purple colour, many of the flowers grew variegated, or virgin white. The researchers concluded that the introduced chalcone synthase gene had somehow muted both itself and a normal petunia gene. Joergensen et al termed this phenomenon of gene silencing "cosuppression" (1).
Their discoveries were supported by another group studying plant RNA viruses. Baulcombe et al (2) were expressing genes from the potato virus X in tobacco plants. The researchers hoped that viral proteins produced by the plants would stimulate its defence allowing the plants to resist subsequent attack by the virus itself. To their surprise the plants with the strongest resistance were those in which the introduced gene was silent. The researchers concluded that the introduced gene was co-suppressing both itself and the same gene in the virus.
In fungi, gene silencing was observed during attempts to boost the production of an orange pigment by the mould Neurospora crassa. Macino and Cogoni introduced extra copies of a gene involved in making a carotenoid pigment. In their experiments a third of the engineered mould bleached out, rather than turning to a deeper orange. Something had suppressed the pigment gene. They termed the observed phenomenon of gene silencing "quelling" (3,4).
Other scientists working with Caenorhabditis elegans obtained strange results in their antisense experiments. The theory behind the antisense approach is to inject complementary RNA sequences into the target organism to block the targeted mRNA. The two sequences should then hybridize stopping the production of the encoded protein. To Guo's surprise even the injected sense strand was active (5). This was later explained as the sense strand used was contaminated with very small amounts of the corresponding antisense strand . In a classic antisense approach these small contaminations would have shown no effect at all.
In 1998 Fire et al., suggested a new mechanism for the phenomenon of gene silencing. In their experiments using Caenorhabditis elegans they showed that double stranded RNA (dsRNA) was even more effective in gene silencing than both sense or antisense strands alone (7). They found that only a few molecules of injected dsRNA were required per affected cell. Fire et al. described this mechanism as extremely gene specific and suggested that the dsRNA mediated silencing was part of a complex biological regulation system. Fire et al. named the phenomenon of gene silencing RNA interference (RNAi).
RNAi Mechanism and Short Interfering RNA (siRNA)
Consistent with gene silencing by dsRNA, Hamilton et al., described the existence of small (about 25nt) RNAs that correspond to the gene that has been silenced in plants .
While looking for a common principle Hammond et al., detected similar short RNAs in Drosophila. They suggested that these are incorporated into a RNA induced silencing complex (RISC) and then are used as a guide in the RNAi mechanism, which then leads to degradation of the corresponding mRNA (9).
Today the basic mechanism of RNA interference (as it has been shown for Drosophila) can be understood as a two step process (10).
First, the dsRNA is cleaved to yield short interfering RNAs (siRNAs) of about 21-23nt length (8, 9, 11-13) with 5' terminal phosphate and 3' short overhangs (~2nt) (12). Then the siRNAs target the corresponding mRNA sequence specific for destruction (Fig. 1) (7-9,11,13,14).
Fig. 1: After dsRNA (of >30nt) is transfered into the cellular system, Dicer (Drosophila; 13) or another RNase III-like enzyme breaks the dsRNA into shorter RNA sequences (about 21-23 nt). These short sequences are called short interfering RNA (siRNA). siRNAs direct target specific mRNA degradation in the RNA induced silencing complex (RISC) (9).
Hammond et al., concluded that the identical size of RNA fragments in plants and animals must be the result of a highly conserved mechanism in nature (9). This theory has been supported by many studies showing that dsRNA induced gene silencing can be found in a number of different species (7, 15-24).
Non-Specific Response of Mammalian Cells
Even though it has been shown that dsRNA can mediate gene-specific interference in early mouse embryos and in mouse oocytes (25, 26), the introduction of dsRNA into somatic mammalian cells is limited. Instead of triggering RNAi, the introduced dsRNA generates a general, non-specific decrease of mRNA often followed by cell death. One response to dsRNA in mammalian cells is mediated by the dsRNA-dependent protein kinase (PKR) which phophorylates and inactivates the translation factor eIF2a, leading to a generalized suppression of protein synthesis, and in some cases apoptosis (27).
siRNA as a Tool
Several recent discoveries have begun to overcome the difficulties of unspecific responses and the cell death of mammalian cells in RNAi experiments.
Elbashir et al., analyzed the rate of 21-23 nt fragment formation after successfully triggering RNAi by several dsRNA in their described Drosophila lysate in vitro system (28). The authors then triggered RNAi efficiently using chemically synthesized siRNA duplexes of the same structure with 3'-overhang ends (11).
In a following study Tuschl et al., demonstrated that chemically synthesized 21 nt siRNA duplexes specifically suppress expression of endogenous and heterologeous genes in different mammalian cell lines, including human kidney (293) and HeLa cells (29). A key discovery of these studies was that no unspecific effects occurred in mammalian cells by transfection of short sequences (<30nt). The authors suggested that 21 nt siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.
Caplen et al., supported these discoveries on siRNAs mediating RNAi in cell extracts and presented data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice (30). This study also presents evidence that siRNAs can have direct effects on gene expression in C. elegans and mammalian cell culture in vivo.
siRNA an Outlook
Today many researchers are excited about the huge potential of siRNA. In Nature Structural Biology Zamore wrote (10): "The ability to initiate RNAi in cultured mammalian cells using siRNA duplexes should dramatically accelerate the pace of reverse genetic analysis of the human genome. It will not be surprising if in five years the loss of function phenotype of virtually every human gene will have been examined in cultured cells using siRNA-mediated RNAi. In fact, new technologies may soon make it possible to fabricate RNAi chips - arrays of siRNAs on which cultured cells of many types can be grown and scored for the effects of suppressing expression of every gene in the genome, one-by-one."
We are commited to focusing our resources on this fast growing field of research. We strongly believe that siRNA technology is the most powerful tool to unravel the function of genes. In combination with our TOM-Chemistry and our expertise in RNA synthesis we hope to contribute significantly to the growth of this exciting technology. In our eyes siRNA will soon be used in a variety of applications such as high throughput target validation and gene therapy.
Literature and further reading
(1) Jorgensen RA., Cluster PD, English J., Que Q., Napoli CA., "Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences", Plant Mol. Biol., 31 (5), 957-73, (1996)
(2) Baulcombe DC., "Fast forward genetics based on virus-induced gene silencing", Curr. Opin. Plant. Biol., 2, 109-113, (1999)
(3) Cogoni C, Irelan JT., Schumacher M., Schmidhauser TJ., Selker EU., Macino G., "Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation", EMBO J., 15 (12), 3153-63, (1996)
(4) Cogoni C., Macino G.,Proc. Natl Acad. Sci. USA 94, 10233-10238, (1997)
(5) Guo S. & Kemphues K., "par-1, a gene required for establishing polarity in embryos, encodes a putative Ser/Thr kinase that is symmetrically disrupted", Cell, 81, 611-620 (1995)
Susan Parrish, Jamie Fleenor, SiQun Xu, Craig Mello and Andrew Fire, "Functional Anatomy of a dsRNA Trigger: Differential Requirement for the Two Trigger Strands in RNA Interference", Molecular Cell, 6, 1077-87, (2000)
(7) Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C., " otent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans", Nature, Vol 391, (1998)
Hamilton AJ. and Baulcombe DC., A species of small antisense RNA in posttranscriptional gene silencing in plants, Science, 286, 950-952, (1999)
(9) Hammond SM., Bernstein E., Beach D., Hannon GJ., An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells., Nature 404, 293-296, (2000)
(10) Zamore P.D., "RNA interference: listening to the sound of silence", Nature Structural Biology, 8, 9, 746-750, (2001)
(11) Zamore PD., Tuschl T., Sharp PA.& Bartel DP., "RNAi: Double stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals", Cell, 101, 25-33, (2000)
(12) Elbashir SM., Lendeckel W. & Tuschl T., "RNA interference is mediated by 21- and 22-nucleotide RNAs", Genes & Development, 15, 188-200, (2001)
(13) Bernstein E., Caudy A.A., Hammond S.M., Hannon G.J., "Role for a bidentate ribonuclease in the initiation step of RNA interference", Nature 409, 363-366, 2001
(14) Yang D., Lu H., Erickson J.W.,"Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos", Curr. Biol., 10, 1191-1200, (2000)
(15) Kennerdell J.R., Carthew R.W., "Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway", Cell, 95, 1017-1026, (1998)
(16) Montgomery M.K., Xu S., Fire A., "RNA as a target of double-stranded RNA-mediated genetic interference in Caenorrhabditis elegans", Proc. Natl. Acad. Sci. USA 95, 15502-15507, (1998)
(17) Ngo H., Tschudi C., Gull K., Ullu E., "Double-stranded RNA induces mRNA degradation in Trypanosoma brucei", Proc. Natl. Acad. Sci. USA 95, 14687-14692, (1998)
(18) Timmons L., Fire A., "Specific interference by ingested dsRNA", Nature 395, 854, (1998)
(19) Bahramian M.B., Zarbl H., "Transcriptional and post-transcriptional silencing of rodent 1 collagen by a homologous transcriptionally self-silenced transgene", Mol. Cell. Biol., 19, 274-283, (1999)
(20) Lohmann J.U., Endl I., Bosch T.C., "Silencing of developmental genes in hydra", Dev. Biol., 214, 211-214, (1999)
(21) Misquitta L., Paterson B.M., "Targeted disruption of gene function in Drosophila by RNA interference (RNA-I): a role for nautilus in embryonic somatic muscle formation", Proc. Natl. Acad. Sci. USA 96, 1451-1456, (1999)
(22) Sanchez A.A., Newmark P.A., "Double-stranded RNA specifically disrupts gene expression during planarian regeneration", Proc. Natl. Acad. Sci. USA 96, 5049-5054, (1999)
(23) Wargelius A., Ellingsen S., Fjose A., "Double-stranded RNA induces specific developmental defects in zebrafish embryos", Biochem. Biophys. Res. Commun., 263, 156-161, (1999)
(24) Li Y.X., Farrell M.J., Liu R., Mohanty N., Kirby M.L., Double-stranded RNA injection produces null phenotypes in zebrafish, Dev. Biol., 217, 394-405, (2000)
(25) Wianny F., Zernicka-Goetz M., Specific interference with gene function by double-stranded RNA in early mouse development, Nature Cell Biol., 2, 70-75, (2000)
(26) Svoboda P., Stein P., Hayashi H. and Schultz R.M.,"Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference", Development (Cambridge, U.K.) 127, 4147-4156, 2000)
(27) Clemens M.J., Elia A., "The double stranded RNA-dependent protein kinase PKR: structure and function", J. Interferon Cytokine Res., 17, 503-524, (1997)
(28) Tuschl T., Zamore PD., Lehmann R., Bartel DP. & Sharp PA., "Targeted mRNA degradation by double-stranded RNA in vitro", Genes & Dev., 13, 3191-3197, (1999)
(29) Elbashir SM. et al., "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells", Nature, 411, 494-498, (2001)
(30) Natasha J. Caplen, Susan Parrish, Farhad Imani, Andrew Fire and Richard A. Morgan, "Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems", PNAS early Edition, 171251798, 1-6, (2001)
(31) Fay D.S., Stanley H.M., Han M., Wood W.B., "A Caenorhabditis elegans homologue of hunchback is required for late stages of development but not early embryonic patterning", Dev. Biol., 205, 240-253, (1999) |
|
发表于 2004-11-23 10:22:02
9NPG2"
