Gene Silencing, RNA interference (RNAi), Virus-Induced Gene Silencing (VIGS)

Gene silencing is a general term describing epigenetic processes of gene regulation. The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic mutation. That is, a gene which would be expressed (turned on) under normal circumstances is switched off by machinery in the cell.

Transcriptional gene silencing is the result of histone modifications, creating an environment of heterochromatin around a gene that makes it inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc.).

Post-transcriptional gene silencing is the result of mRNA of a particular gene being destroyed. The destruction of the mRNA prevents translation to form an active gene product (in most cases, a protein). A common mechanism of post-transcriptional gene silencing is RNAi (RNA interference), see below.

Both transcriptional and post-transcriptional gene silencing are used to regulate endogenous genes. Mechanisms of gene silencing also protect the organism's genome from transposons and viruses. Gene silencing thus may be part of an ancient immune system protecting from such infectious DNA elements.

Post transcriptional gene silencing (PTGS) is a mechanism for sequence-specific RNA degradation in plants.

The process was described first in transgenic Petunia. The scientists’ goal was to produce petunia plants with improved flower colors. To achieve this goal, they introduced additional copies of a gene encoding a key enzyme for flower pigmentation into petunia plants. Surprisingly, many of the petunia plants carrying additional copies of this gene did not show the expected deep purple or deep red flowers but carried fully white or partially white flowers. When the scientists had a closer look they discovered that both types of genes, the endogenous and the newly introduced transgenes, had been turned off. Because of this observation the phenomenon was first named “co-suppression of gene expression” but the molecular mechanism remained unknown.

A few years later plant virologists made a similar observation. In their research they aimed towards improvement of resistance of plants against plant viruses. At that time it was known that plants expressing virus-specific proteins show enhanced tolerance or even resistance against virus infection. However, they also made the surprising observation that plants carrying only short regions of viral RNA sequences not coding for any viral protein showed the same effect. They concluded that viral RNA produced by transgenes can also attack incoming viruses and stop them from multiplying and spreading throughout the plant. They did the reverse experiment and put short pieces of plant gene sequences into plant viruses. Indeed, after infection of plants with these modified viruses the expression of the targeted plant gene was suppressed. They called this phenomenon “virus-induced gene silencing” or simply “VIGS”.

The ability of RNAi to dramatically and selectively reduce the expression of an individual protein in a cell makes RNAi a valuable laboratory research tool, both in cell culture and in vivo in living organisms. It is particularly useful in certain organisms such as C. elegans, in which the gene silencing phenotype is heritable, in Drosophila, and in plants where the effect can spread from cell to cell within the organism. Large-scale screens that systematically shut down each protein in the cell can aid in identifying the necessary components for a particular cellular process or event and can identify which proteins are required for cell survival and replication. RNAi also holds promise as a therapeutic technique in human disease.

Cellular mechanism

RNAi is an RNA-dependent gene silencing process that is mediated by the same cellular machinery that processes microRNA, known as the RNA-induced silencing complex (RISC). The process is initiated by the ribonuclease protein Dicer,^[4] which binds and cleaves exogenous double-stranded RNA molecules to produce double-stranded fragments of 20-25 base pairs with a few unpaired overhang bases on each end.^[5] The short double-stranded fragments produced by Dicer, called small interfering RNAs (siRNAs), are separated and integrated into the active RISC complex. Although it was first believed that an ATP-dependent helicase separated the two strands,^[6] it has since been shown that the process is ATP-independent and effected directly by the protein components of RISC.^[7]

The catalytically active components of the RISC complex are known in animals as argonaute proteins, endonucleases which mediate the siRNA-induced cleavage of the target mRNA strand. Because the fragments produced by Dicer are double-stranded, they could each in theory produce a functional siRNA; however, only one of the two strands - known as the guide strand - binds the argonaute protein and leads to gene silencing. The other anti-guide strand or passenger strand is degraded as a RISC substrate during the process of RISC activation.^[8] The strand selected as the guide tends to be the strand whose 5' end is more stable, but strand selection is not dependent on the direction in which Dicer cleaves the dsRNA before RISC incorporation.^[9]

It is not yet well understood how the activated RISC complex locates complementary mRNA molecules within the cell. Although the cleavage process has been proposed to be linked to translation, it has been shown that translation of the mRNA target is not a prerequisite for RNAi-mediated degradation.^[10] In fact, one study found an increase in RNAi activity against mRNA targets that were not translated.^[11] Argonaute proteins, the catalytic components of RISC, have been identified as localized to specific regions in the cytoplasm called cytoplasmic bodies, which are also local regions of high mRNA decay rates.^[12]

The native cellular purpose of the RNA interference machinery is not well characterized, but it is known to be involved in microRNA (miRNAs) processing and the resulting translational repression. MicroRNAs, which are encoded in the genome and have a role in gene regulation, typically have incomplete base pairing and only inhibit the translation of the target mRNA; by contrast, RNA interference as used in the laboratory typically involves perfectly base-paired dsRNA molecules that induce mRNA cleavage. ^[13] After integration into the RISC, siRNAs base pair to their target mRNA and induce the RISC component protein argonaute to cleave the mRNA, thereby preventing it from being used as a translation template.

Organisms vary in their cells' ability to take up foreign dsRNA and use it in the RNAi pathway. The effects of RNA interference are both systemic and heritable in plants and in C. elegans, although not in Drosophila or mammals due to the absence of RNA replicase in these organisms. In plants, RNAi is thought to propagate through cells via the transfer of siRNAs through plasmodesmata.^[6]

Biological origins

The RNA interference pathway is thought to play a role in the immune response to viruses and other foreign genetic material, especially in plants where it may also protect against the self-propagation of transposons.^[14] The pathway is conserved across all eukaryotes, although it has been independently recruited to play other functions such as histone modification,^[15] the reorganization of genomic regions with complementary sequence to induce heterochromatin formation,^[16] and maintenance of centromeric heterochromatin.^[17]

For miRNA's, certain parts of the genome are transcribed into short RNA molecules that fold back on themselves in a hairpin shape to create a double strand primary miRNA structure (pri-miRNA). The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release the mature miRNA. If base-pairing with the target is perfect or near-perfect this may result in cleavage of messenger RNA (mRNA). This is quite similar to the siRNA function, however, many miRNA's will base pair with mRNA with an imperfect match. In such cases, the miRNA causes the inhibition of translation and prevents normal function. Consequently, the RNAi machinery is important to regulate endogenous gene activity. This effect was first described for the worm Caenorhabditis elegans in 1993 by R. C. Lee et al. of Harvard University.^[18] In plants, this mechanism was first shown in the "JAW microRNA" of Arabidopsis; it is involved in the regulation of several genes that control the plant's shape.^[19] Genes have been found in bacteria that are similar in the sense that they control mRNA abundance or translation by binding an mRNA by base pairing, however they are not generally considered to be miRNA's because the Dicer enzyme is not involved.^[20] It has been suggested that CRISPR systems in prokaryotes are analogous to eukaryotic RNA interference systems, although none of the protein components is orthologous. ^[21]

History

The revolutionary finding of RNAi resulted from the unexpected outcome of experiments performed by plant scientists in the USA and The Netherlands.^[22] The goal was to produce petunia plants with improved flower colors. To achieve this goal, they introduced additional copies of a gene encoding a key enzyme for flower pigmentation into petunia plants. Surprisingly, many of the petunia plants carrying additional copies of this gene did not show the expected deep purple or deep red flowers but carried fully white or partially white flowers. When the scientists had a closer look they discovered that both types of genes, the endogenous and the newly introduced transgenes, had been turned off. Because of this observation the phenomenon was first named "co-suppression of gene expression" but the molecular mechanism remained unknown.

A few years later plant virologists made a similar observation. In their research they aimed towards improvement of resistance of plants against plant viruses. At that time it was known that plants expressing virus-specific proteins show enhanced tolerance or even resistance against virus infection. However, they also made the surprising observation that plants carrying only short regions of viral RNA sequences not coding for any viral protein showed the same effect. They concluded that viral RNA produced by transgenes can also attack incoming viruses and stop them from multiplying and spreading throughout the plant. They did the reverse experiment and put short pieces of plant gene sequences into plant viruses. Indeed, after infection of plants with these modified viruses the expression of the targeted plant gene was suppressed. They called this phenomenon “virus-induced gene silencing” or simply “VIGS”. These phenomena are collectively called post transcriptional gene silencing.

After these initial observations in plants many laboratories around the world searched for the occurrence of this phenomenon in other organisms. Mello and Fire's 1998 Nature paper based on research conducted with their colleagues (SiQun Xu, Mary Montgomery, Stephen Kostas, Sam Driver) at the Carnegie Institution of Washington and the University of Massachusetts reported a potent gene silencing effect after injecting double stranded RNA into C. elegans.^[2] In investigating the regulation of muscle protein production, they observed that neither mRNA and antisense RNA injections had an effect on protein production, but double-stranded RNA successfully silenced the targeted gene. As a result of this work, they coined the term RNAi. The discovery of RNAi in C. elegans is particularly notable, as it represented the first identification of the causative agent (double stranded RNA) of this heretofore inexplicable phenomenon. Fire and Mello were awarded the Nobel Prize in Physiology or Medicine in 2006 for their work.

Gene knockdown

RNAi has recently been applied as an experimental technique to study the function of genes in model organisms. Double-stranded RNA for a gene of interest is introduced into a cell or organism, where it through RNAi causes an often drastic decrease in production of the protein the gene codes for. Studying the effects of this decrease can yield insights into the protein's role and function. Since RNAi may not totally abolish expression of the gene, this technique is sometimes referred as a "knockdown", to distinguish it from "knockout" procedures in which expression of a gene is entirely eliminated by removing or destroying its DNA sequence.

Most functional genomics applications of RNAi have used the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, both commonly used model organisms in genetics research.^[23] C. elegans is particularly useful for RNAi research because the effects of the gene silencing are generally heritable and because delivery of the dsRNA is exceptionally easy. Via a mechanism whose details are poorly understood, bacteria such as E. coli that carry the desired dsRNA can be fed to the worms and will transfer their RNA payload to the worm via the intestinal tract. This "delivery by feeding" yields essentially the same magnitude of gene silencing as do more costly and time-consuming traditional delivery methods, such as soaking the worms in dsRNA solution and injecting dsRNA into the gonads.^[24]

Role in medicine

It may be possible to exploit the RNA interference process for therapeutic purposes. Although it is difficult to introduce long dsRNA strands into mammalian cells due to the interferon response, the use of short interfering RNA mimics has been more successful.^[25] The first applications to reach clinical trials are in the treatment of macular degeneration and respiratory syncytial virus.^[26] RNAi has also been shown effective in the complete reversal of induced liver failure in mouse models.^[27]

Other proposed clinical uses explored in cell culture center on antiviral therapies, including the inhibition of viral gene expression in cancerous cells,^[28] the silencing of hepatitis A^[29] and hepatitis B^[30] genes, silencing of influenza gene expression,^[31] and inhibition of measles viral replication.^[32] Potential treatments for neurodegenerative diseases have also been proposed, with particular attention to the polyglutamine diseases such as Huntington's disease.^[33]

Despite the proliferation of promising cell culture studies for RNAi-based drugs, some concern has been raised regarding the safety of RNA interference, especially the potential for "off-target" effects in which a gene with a coincidentally similar sequence to the targeted gene is also repressed.^[34] A computational genomics study estimated that the error rate of off-target interactions is about 10%.^[35] One major study of liver disease in mice led to high death rates in the experimental animals, suggested by researchers to be the result of "oversaturation" of the dsRNA pathway.^[36]

References

^ Nobel prize for genetic discovery. BBC (2006-10-02). Retrieved on 2006-10-02.
^ ^a ^b A. Fire, S.Q. Xu, M.K. Montgomery, S.A. Kostas, S. E. Driver, C.C. Mello: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. In: Nature. 391/1998, S. 806-811, ISSN 0028-0836
^ Macrae IJ, Zhou K, Li F, Repic A, Brooks AN, Cande WZ, Adams PD, Doudna JA. (2006). Structural basis for double-stranded RNA processing by Dicer. Science 311(5758):195-8.
^ Bernstein E, Caudy AA, Hammond SM, Hannon GJ. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409(6818):363-6.
^ Vermeulen A, Behlen L, Reynolds A, Wolfson A, Marshall WS, Karpilow J, Khvorova A. (2005). The contributions of dsRNA structure to Dicer specificity and efficiency. RNA 11(5):674-82.
^ ^a ^b Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipurksy SL, Darnell J. (2004). Molecular Cell Biology 5th ed. WH Freeman: New York, NY.
^ Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD. (2005). Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123(4):607-20.
^ Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R. (2005). Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123(4):631-40.
^ Preall JB, He Z, Gorra JM, Sontheimer EJ. (2006). Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila. Curr Biol 16(5):530-5.
^ Sen GL, Wehrman TS, Blau HM. (2005). mRNA translation is not a prerequisite for small interfering RNA-mediated mRNA cleavage. Differentiation 73(6):287-93.
^ Gu S, Rossi JJ. (2005). Uncoupling of RNAi from active translation in mammalian cells. RNA 11(1):38-44.
^ Sen GL, Blau HM. (2005). Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat Cell Biol 7(6):633-6.
^ Schramke V, Allshire R. (2003). Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing. Science 301(5636):1069-74. PMID 12869699
^ Stram Y, Kuzntzova L. (2006). Inhibition of viruses by RNA interference. Virus Genes 32(3):299-306.
^ Cerutti H, Casas-Mollano JA. (2006). On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet 50(2):81-99.
^ Holmquist GP, Ashley T. (2006). Chromosome organization and chromatin modification: influence on genome function and evolution. Cytogenet Genome Res 114(2):96-125.
^ Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA. (2002). Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297(5588):1833-7. PMID 12193640
^ Lee RC, Feinbaum RL, Ambros V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843-54.
^ Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D. (2003). Control of leaf morphogenesis by microRNAs. Nature 425(6955):257-63.
^ Morita T, Mochizuki Y, Aiba H. (2006). Translational repression is sufficient for gene silencing by bacterial small noncoding RNAs in the absence of mRNA destruction. Proc Natl Acad Sci 103(13):4858-63.
^ Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. (2006). A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 16;1:7. PMID: 16545108
^ Napoli C., Lemieux C., and Jorgensen R. (1990) "Introduction of a chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans". Plant Cell 2: 279-289.
^ Dzitoyeva S, Dimitrijevic N, Manev H. (2003). Gamma-aminobutyric acid B receptor 1 mediates behavior-impairing actions of alcohol in Drosophila: adult RNA interference and pharmacological evidence. Proc Natl Acad Sci 100(9):5485-90. PMID 12692303
^ Fortunato A, Fraser AG. (2005). Uncover genetic interactions in Caenorhabditis elegans by RNA interference. Biosci Rep 25(5-6):299-307. PMID 16307378
^ Paddison PJ, Caudy AA, Hannon GJ. (2002). Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci USA 99(3):1443-8.
^ Sah DW. (2006). Therapeutic potential of RNA interference for neurological disorders. Life Sci Epub.
^ Zender L, Hutker S, Liedtke C, Tillmann HL, Zender S, Mundt B, Waltemathe M, Gosling T, Flemming P, Malek NP, Trautwein C, Manns MP, Kuhnel F, Kubicka S. (2003). Caspase 8 small interfering RNA prevents acute liver failure in mice. Proc Natl Acad Sci USA 100(13):7797-802.
^ Jiang M, Milner J. (2002). Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene 21(39):6041-8.
^ Kusov Y, Kanda T, Palmenberg A, Sgro JY, Gauss-Muller V. (2006). Silencing of hepatitis A virus infection by small interfering RNAs. J Virol 80(11):5599-610.
^ Jia F, Zhang YZ, Liu CM. (2006). A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference. Biotechnol Lett 28(20):1679-85.
^ Li YC, Kong LH, Cheng BZ, Li KS. (2005). Construction of influenza virus siRNA expression vectors and their inhibitory effects on multiplication of influenza virus. Avian Dis 49(4):562-73.
^ Hu L, Wang Z, Hu C, Liu X, Yao L, Li W, Qi Y. (2005). Inhibition of Measles virus multiplication in cell culture by RNA interference. Acta Virol 49(4):227-34.
^ Raoul C, Barker SD, Aebischer P. (2006). Viral-based modelling and correction of neurodegenerative diseases by RNA interference. Gene Ther 13(6):487-95.
^ Tong AW, Zhang YA, Nemunaitis J. (2005). Small interfering RNA for experimental cancer therapy. Curr Opin Mol Ther 7(2):114-24.
^ Qiu S, Adema CM, Lane T. (2005). A computational study of off-target effects of RNA interference. Nucleic Acids Res 33(6):1834-47.
^ Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA. (2006). Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441(7092):537-41.

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