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Gene Silencing by RNA Interference and the Role of Small Interfering RNAs
Natasha J. Caplen
1.1 The Identification of Post-Transcriptional Gene-Silencing Mechanisms 1.2 RNA Silencing, siRNAs, and RISC 1.3 Species-Specific Aspects of RNA-Triggered Gene Silencing 1.4 RNAi and Epigenetic Endogenous RNA Silencing 1.5 Applications of RNAi-Based Technologies References
The completion or near completion of several whole genome sequence projects has highlighted two important issues: (1) a far higher percentage of transcription results in the generation of non-coding RNAS than was originally predicted, and (2) there is now a need to rapidly translate genomic information into functional data. The recent identification of RNA interference (RNAi) an endogenous post-transcriptional genesilencing mechanism that requires formation of a non-coding RNA containing protein complex, and the rapid development of technologies that exploit this mechanism to determine gene function illustrates the need to characterize basic cellular processes so that these can be exploited as scientific tools. This chapter summarizes RNA-mediated epigenetic gene-silencing mechanisms, the identification and role of key players in RNAi and how this work has impacted on both our understanding of the role of noncoding RNAs in regulating endogenous gene expression and on the development of a whole new research field utilizing RNAi-generated knockdowns, even up to a genomewide scale to study gene function.
1.1 THE IDENTIFICATION OF POST-TRANSCRIPTIONAL GENE SILENCING MECHANISMS
Post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) has been observed in a wide range of organisms including plants, with Nicotina and Arabidopsis the species most widely studied; fungi, in particular Neurospora crassa; several
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20.18 Central to PTGS and RNAi in all species studied to date is the role of small RNA molecules (20 to 25 nts in length) and the formation of a ribonucleoprotein complex called the RNA-induced silencing complex or RISC.24. RNA silencing can be observed following the introduction of multiple copies of a transgene and in cells infected with cytoplasmically replicating RNA viruses. The first evidence suggesting a role for small RNA molecules in PTGS and RNAi emerged from a plant study that showed that in cells where PTGS had been triggered (by a transgene or virus) there was an accumulation of a small species of RNA estimated to be approximately 25 nucleotides (nts) in size.22.27–29 Cleavage of the target mRNA was observed at a site that corresponded to approximately the middle of the siRNA.2 Gene Silencing by RNA Interference Metazoan and Protozoan species.2 RNA SILENCING.26 Double-stranded RNA was quickly shown to mediate a similar inhibition of gene expression in other invertebrate species.30 Since the identification of siRNAs. AND RISC siRNAs processed from dsRNA within a cell are typically of 20 to 25 nts in length. elegans is highly potent. are enzymatically derived from the input doublestranded RNA. suggesting that an intracellular process both amplifies and spreads this effect. it was unclear for some time how degradation of the target RNA was mediated.29 The siRNAs are duplex RNA molecules with 2 or 3 nt 3' overhangs of ssRNA and carry a Copyright 2005 by CRC Press LLC . or introduced exogenously appeared to be the triggers of both PTGS and RNAi. including Drosophila.25 The term RNAi was first used to describe the inhibition of gene expression seen in C. SIRNAS. now termed small or short interfering RNAs (siRNAs).6. elegans following the direct injection into embryos of double-stranded RNA (dsRNA) of a sequence cognate to the gene that was silenced. respectively).9 Though dsRNA generated through aberrant transcription from multiple transgenes (particularly those arranged as an inverted repeat). or from a viral intermediate. Drosophila.23 Plants defective for PTGS. and most recently mammalian cells both human and mouse.1–17 The term PTGS has been used mostly to indicate epigenetic post-transcriptional gene-silencing in plants and fungi (also known as co-suppression and quelling. show increased susceptibility to infection and viruses produce proteins that block PTGS.19 Subsequent in vitro analysis of RNAi in Drosophila cell extracts showed that a small species of RNA. leading to the hypothesis that PTGS acts as an antiviral response in plants. and more complex organisms. particularly Caenorhabditis elegans. and Trypanosoma. suggesting that the siRNA acts as guide for an enzyme-containing complex that leads to degradation of the target mRNA. though it has been suggested that the collective term of RNA silencing is more appropriate for all of these processes. their central role in PTGS and RNAi has been more fully defined and their relationship to endogenous small non-coding RNAs has begun to be elucidated.22.22 The enzyme responsible for the processing of siRNAs from dsRNA is an RNase III-like endonuclease termed Dicer.19–22 In plants. for example. 1.7. Planarian. Hydra.20. though most have been sized as 21 to 23 nt.8.6 As in plants and fungi the silencing induced by dsRNA in C. whereas RNAi has been mostly applied to a similar epigenetic gene-silencing effect seen in invertebrates and mammals.
20. can also bind.31–33 siRNAs can be generated from all parts of the input dsRNA but there may be an accumulation of those sequences that most effectively induce silencing. but the presence of a 3' singlestranded RNA overhang appears to be critical to the interactions. Many viruses produce dsRNA intermediates during their life cycle. is involved in the transfer of siRNAs as they are generated by Dicer onto proteins that from part of the RISC complex. Argonaute 2 (Ago2) appears to form the core of RSIC.31. the Vasa intronic gene product (a p68 RNA helicase homolog) and the fragile X mental retardation protein.28 It is unclear at exactly what point an individual siRNA is unwound. ssRNA can enter the RNAi pathway but do so fair less efficiently.34 The internal free energy around the predicted cleavage site also appears to need to be relatively low in comparison with adjacent regions of the molecule.40. but stable RISC complexes appear to contain just one strand of the siRNA.Gene Silencing by RNA Interference and the Role of Small Interfering RNAs 3 5' phosphate group. it is assumed that this allows the siRNA to be efficiently unwound.38.and double-stranded. Argonaute.41 The affinity of the interaction of this domain and RNA is relatively weak.36 R2D2 may influence the polarity (sense/antisense orientation) with which the siRNA interacts with components of RISC. The best-characterized responses involve the triggering RNase L-dependent pathways through activation of 2'5'-oligoadenylate Copyright 2005 by CRC Press LLC .22.41 RNA. single.34. and thus as part of an antiviral response mammalian cells have evolved potent responses to dsRNA that result in a nonspecific down-regulation in gene expression.42–46 The antisense single-stranded siRNA component of RISC leads to localization of the RISC complex through perfect sequence alignment of the two RNA molecules. called R2D2. The first to be identified.41 Other proteins associated with RISC include in Drosophila. and in human cells the homolog of Ago2–E2FC and Gemin3 and Gemin4.34 There is evidence from studies of RNAi in Drosophila cells that at least one protein.3 SPECIES-SPECIFIC ASPECTS OF RNA-TRIGGERED GENE SILENCING The identification of siRNAs as intermediate molecules in the RNAi pathway was essential to establishing the presence of RNAi in mammalian cells. and Zwille/Pinhead) domain-containing family of proteins. Several protein components of RISC have been isolated.28 The ribonuclease(s) responsible for this cleavage is not defined but an enzyme with homology to Tudor staphylococcal nuclease is associated with RISC and effective RNAi.39 Recent structural analysis of the Drosophila Ago2 protein has shown that the PAZ domain can form a nucleic acid-binding fold. Cleavage of the target occurs at a position ~10 nts from the first nucleotide that represented the first based pair from the 5' end of the original siRNA.40. though in the case of duplex DNA the binding is independent of a 3' overhang.37 Ago2 and Dicer are both members of the PAZ (PIWI.35 This selection may relate to certain features of the sequence of siRNAs that influence the effectiveness of a particular siRNA. An important feature may be the internal energy of the siRNA molecule.20. which in most effective siRNAs seems to be lower than other portions of the molecule. a process that is ATP dependent. particularly the internal stability of the 5' end of the antisense strand compared to the target sequences.47 1.
but in the vast majority of studies molecules are used based on a siRNA structure.48.64 An early and important observation unique to gene silencing in plants. as this amplification process is termed. crassa (QDE-1) and C. To test if RNA molecules modeled on the structure of siRNAs could mediate sequence-specific cleavage in mammalian cells. one of ~21 to 22 nts in length and another group of ~24 to 26 nts that are probably generated by different Dicer enzymes. usually from RNA polymerase III promoters. however. Neurospora and C. Double-stranded RNAs of over 30 nts can be used in cells that have an attenuated response to larger dsRNAs such as those derived from embryonic cells. but candidates include siRNAs. N. with one model suggesting that short siRNAs can move directly to ~10 to 15 cells but for more extensive spread synthesis of additional siRNAs is required.22 In the last two years these findings have been utilized and extended by many groups using cell lines derived from a very broad range of cells and against transcripts involved in a broad range of cellular processes. larger dsRNAs. elegans (Ego-1. non-specific effects have been reported that may reflect some low level triggering of non-RNAi dsRNA dependent pathways.70 The 21 to 22 nt siRNAs appear to be critical for mediating localized RNA silencing. The local (cell-to-cell plasmodesmata-based movement) and distant (phloem-based transport) spread of RNA silencing seen in plants can be observed following the use of an exogenous nucleic acid trigger and transgenic plants that induce RNA silencing in grafts. enabling the generation of cell lines expressing a shRNA against a particular transcript.50–56 Mammalian cells show no amplification or spread of the inhibition of gene expression mediated by synthetic siRNAs.69.57–59 To overcome this limitation when applying RNAi as a functional genomics tool in mammalian cells.16.22 Endogenous gene expression could also be downregulated using siRNAs against Lamin A/C.4 Gene Silencing by RNA Interference synthetase and the interferon-associated dsRNA-dependent kinase (PKR) but there is also evidence of additional dsRNA-sensitive pathways. and an RNA/protein complex.63.54. two groups transfected siRNA molecules made from chemically synthesized oligoribonucleotides into a variety of different cell lines.22 Initially. RRF-1).16.61.68 Recent data suggests that plants generate two different species of siRNA.60–64 A key advantage of these short hairpin RNAs (shRNAs) is that they can be expressed from plasmids. concentration-dependent saturation of RISC which may effect its cellular role. elegans is the amplification and spread of the gene-silencing effect. many groups have developed siRNAs that are expressed as a single transcript that forms a stem–loop structure where the stem corresponds to the siRNA.72–76 It is presumed that transitive RNA silencing or transitive RNAi.65–67 The molecule(s) that mediate this spread have not been formally identified. SGS2/SDE1).30.49 In most cases the expression of gene expression has been determined to be specific. and off-target effects due to imperfect sequence matches that can trigger alternative RNA silencing mechanisms.31.71 The intracellular generation of secondary siRNAs through the activity of an RNA-dependent RNA polymerase (RdRp) has been identified in plants (Arabidopsis. using a plasmid/siRNA co-transfection model system of RNAi sequence-specific inhibition was detected at both RNA and protein level. Though only limited data exist maximal activation of these pathways may be dependent on the size and concentration of the triggering dsRNA molecule. uses the homologous ssRNA transcript as a template to produce additional dsRNA Copyright 2005 by CRC Press LLC .
whereas another study observed the generation of only the longer species of siRNA. The role of the larger siRNAs is unclear but in one study these have been linked to expression from endogenous retrotransposons that resulted in systemic and stable silencing and it has been suggested that it is this species that could act as a mobile silencing signal through the vascular (phloem) system of plants that could then trigger the intracellular production of small siRNAs at a distant site.30.76. In plants. though one study has reported synthesis of dsRNA in Drosophila cell extracts. there is also evidence for the active transport of dsRNA through the transmembrane protein SID-1 which was identified by analysis of C.82.91–94 Precursor miRNAs are themselves processed from the initially expressed transcript Copyright 2005 by CRC Press LLC . transcriptional or translational level.79–81 Expression of SID-1 as a transgene in Drosophila cells can induce these cells to take up dsRNA from medium that can then mediate RNAi.71 Additional evidence that the systemic spread of RNA silencing in plants could be mediated in different ways depending on the nature of the initiating molecule comes from data using grafting models that showed a difference in the induction of distant RNA-silencing effects depending if the trigger was transgene or amplicon derived. elegans not only is there evidence for the presence of an RdRp-dependent amplification that generates secondary siRNAs.75.77 In Neurospora the QDE-1 RdRp catalyzes at least two activities that could relate to the mediation of RNA silencing. the nature of the siRNA generated following amplification is unclear.81 Drosophila cells naturally show no cell-to-cell movement of RNA silencing and most studies suggest that there is no RdRP amplification of RNAi in Drosophila. In addition. however. with the identification of RNA silencing the role of RNA both as a mediator and as a target for controlling expression has become increasingly apparent. The first evidence for a direct link between RNAi and the control of endogenous gene expression came with the identification of a role for Dicer in the processing what are now known to be an important group of endogenous non-coding RNAs termed micro RNAS (miRNAs).83 1.70.84–90 Micro RNAs are 21–23 nt ssRNA molecules that are processed by Dicer from large stemloop single-stranded RNAs termed precursor miRNAs in an asymmetric manner. elegans mutants that were sensitive to local RNAi but that were defective for systemic RNAi. One study suggests that only siRNAs of 21 nts are produced. there is a need for the cell to protect itself from the potential effect of aberrant expression from the large amount of selfish genetic material that is present within the genome.4 RNAI AND EPIGENETIC ENDOGENOUS RNA SILENCING A highly coordinated control of gene expression is essential for normal development and differentiation and for the maintenance of appropriate cellular function. and shorter < 21 nt ssRNA molecules corresponding to different parts of the template that may interact directly with RISC.69. the generation of full length RNA duplexes from ssRNA templates (with no requirement for a primer) that could then act as substrate for Dicer.Gene Silencing by RNA Interference and the Role of Small Interfering RNAs 5 that is then subjected to Dicer to generate further siRNA. Until recently most research on the control of gene expression has focused on gene regulation at a genomic.71 This difference may reflect species or model systemspecific variations.78 In C.
In plants methylation of genomic DNA is often seen within the transcribed region that encodes the transcript that is the subject of RNA silencing.114.110 This has been best studied when an RNA virus carrying a sequence homologous to the endogenous transcript triggers RNA silencing. if not all. however. In C. This suggests that here either an siRNA-like cleavage or an miRNA-like protein translational blockade could occur.117 Another set of repeat sequences that may be silenced endogenously by RNAi is selfish genetic material such as integrated retroviral and retrotransposons. and RdRp knockout mutant yeast. of the same components of the RISC complex that siRNAs interact with. miRNA-RISC complexes appear to mediate silencing by a somewhat different manner in that the interaction between miRNAs and the putative target transcripts seems to require only minimal homology between the sequences of the two RNA species that leads to a structural perturbation of the mRNA and/or a locking of the RISC complex at the site of binding so that protein translation is blocked.102–105 Very few target transcripts that miRNAs regulate are known. Determining the function of a gene through knockout transgenics has been of enormous value but this has not been applicable on a high throughput basis and is often restricted by aspects of the Copyright 2005 by CRC Press LLC .5 APPLICATIONS OF RNAI BASED TECHNOLOGIES Sequencing and gene expression studies on a whole genome scale have generated enormous amounts of data that imply a function for a particular gene. but a direct functional analysis on a similar scale has not been possible. but of those that have been characterized many are associated with developmentally regulated genes which may explain.86.118–120 1. leading to disruption of both mitosis and meiosis.114.105–108 A second role for RNAi in regulating endogenous expression links it to modifications of genomic DNA that may induce repression of transcription from a variety of sources including coding sequences.31. show transcription from previously silent regions as a result of the loss of histone H3 lysine methylation.97–101 The action of miRNAs may be different in plants in that the miRNAs in plants have exact sequence matches with their putative target mRNAs.114.116 Loss of heterochromatin RNAi in S.115 Overlapping non-coding RNAs expressed from both strands of the repeat sequences within heterochromatin regions have been purified and siRNAs corresponding to these repeat regions have been identified.70. it is unclear how the RNA–DNA interaction triggers methylation or which methylase is involved. which allows for Swi6 localization and thus the spreading of heterochromatin formation. pombe transcribed long terminal repeats can induce RNAi-dependent chromatin silencing that can extend to closely neighboring transcribed sequence. the embryonic lethal phenotype seen in Dicer transgenic knockout mice.111–113 A link between heterochromatin formation and RNAi has currently only been studied in Schizosaccharomyces pombe where Dcr (Dicer). elegans some RNAi mutants show enhanced activation of transposition and in S.42–46 However.109.96 Micro RNAs also interact with many. Ago2.6 Gene Silencing by RNA Interference by another PAZ domain containing RNase III nuclease called Drosha.95. heterochromatin regions and other repetitive sequences including transposons. at least in part. pombe leads to disruption in centromere and telomere function.
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