This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Agrawal, N. Articles by Mukherjee, S. K. Search for Related Content PubMed PubMed Citation Articles by Agrawal, N. Articles by Mukherjee, S. K.

RNA Interference: Biology, Mechanism, and Applications

International Center for Genetic Engineering and Biotechnology, New Delhi 110 067, India

SUMMARY

INTRODUCTION

UNRAVELING RNA SILENCING

PTGS in Plants

Quelling and RNAi

Insights from Virus-Infected Plants (Virus-Induced Gene Silencing)

IMPORTANT FEATURES OF RNA SILENCING

siRNA

Amplification and Systemic Transmission

COMPONENTS OF GENE SILENCING

Dicer

Guide RNAs and RNA-Induced Silencing Complex

RNA and DNA Helicases

Translation Initiation Factor

RNA-Dependent RNA Polymerase

Transmembrane Protein (Channel or Receptor)

Genetic Mutations with Unknown Function

MECHANISM OF RNA INTERFERENCE

Processing of dsRNA into siRNAs

Amplification of siRNAs

Degradation of mRNA

RNA SILENCING FOR GENOME INTEGRITY AND DEFENSE

MECHANISTIC DIFFERENCES AMONG THE BIOSYNTHETIC PATHWAYS OF siRNA

Pre-Dicer stage

Dicer stage

Post-Dicer stage

siRNA: SYNTHESIS, DELIVERY, AND GENE KNOCKDOWN

Selection and Generation of siRNA

Transfection of siRNA and Detection of Gene Silencing

siRNA Introduction into Plants

MICRO-RNA

Identification and Biogenesis

Apoptosis-Related Micro-RNA

Kinship of siRNA- and Micro-RNA-Related Pathways

Functional Classifications

Genetic Diversity in Species-Specific Biosynthesis of Micro-RNA

SMALL-RNA-MEDIATED EFFECTS ON CHROMOSOMAL DNA

RNA-Dependent DNA Methylation

Heterochromatin Formation

DNA Elimination

APPLICATIONS OF RNAi

CONCLUDING REMARKS

REFERENCES

Top Next References

INTRODUCTION

Top Previous Next References

 
RNA silencing is a novel gene regulatory mechanism that limits the transcript level by either suppressing transcription (transcriptional gene silencing [TGS]) or by activating a sequence-specific RNA degradation process (posttranscriptional gene silencing [PTGS]/RNA interference [RNAi]). Although there is a mechanistic connection between TGS and PTGS, TGS is an emerging field while PTGS is undergoing an explosion in its information content. Here, we have limited our discussion to PTGS/RNAi-related phenomena.

Pioneering observations on PTGS/RNAi were reported in plants, but later on RNAi-related events were described in almost all eukaryotic organisms, including protozoa, flies, nematodes, insects, parasites, and mouse and human cell lines, as shown in Table 1. Three phenotypically different but mechanistically similar forms of RNAi, cosuppression or PTGS in plants, quelling in fungi, and RNAi in the animal kingdom, have been described. More recently, micro-RNA formation, heterochromatinization, etc., have been revealed as other facets of naturally occurring RNAi processes of eukaryotic cells.

The natural functions of RNAi and its related processes seem to be protection of the genome against invasion by mobile genetic elements such as viruses and transposons as well as orchestrated functioning of the developmental programs of eukaryotic organisms. There are several excellent recent reviews which deal with different aspects of RNAi separately (95, 191). Here, we have put together the various aspects of the RNAi process known to date, identified the mechanistic similarities and differences operating in various forms of eukaryotic life, and focused on the experimental results that have led to conceptual advancements in this field.

UNRAVELING RNA SILENCING

Top Previous Next References

 
In order to understand the process of homology-dependent RNA silencing, it would be prudent to overview the process itself and describe its important features. In the later part of this review, the genetics, biochemistry, and potential therapeutic applications of the process will be dealt with.

Around the same time, two other laboratories (105, 217) also reported that introduction of the transcribing-sense transgenes could downregulate the expression of homologous endogenous genes. Subsequently, many similar events of cosuppression were reported in the literature. All cases of cosuppression resulted in the degradation of endogene and transgene RNAs after nuclear transcription had occurred (120). Since posttranscriptional RNA degradation was observed in a wide range of transgenes expressing the plant, bacterial, or viral sequences, it was rechristened posttranscriptional gene silencing (PTGS). PTGS could be initiated not only by sense transgenes but also by antisense transgenes, and biochemical evidence suggests that similar mechanisms might operate in both cases (81). It is worthwhile to point out that although the cosuppression phenomenon was originally observed in plants, it is not restricted to plants and has also been demonstrated in metazoans and mammals (98).

In keeping with the times, the observed alterations in the PTGS-related phenotypes were attributed to multiple-site integrations, aberrant RNA formations, repeat structures of the transgenes, etc. Later on, it became clear that the expression of the transgene led to the formation of dsRNA, which, in turn, initiated PTGS. For example, in the case of cosuppressed petunia plants, chsA mRNA formed a partial duplex, since there are regions of self-complementarity located between chsA 3' coding region and its 3' untranslated region (154). This was revealed by DNA sequence analysis and experimental detection of in vitro-transcribed, RNase-resistant duplex chsA RNA. In an independent study, a p35S-ACC (1-aminocyclopropane-1-carboxylate [ACC] oxidase) sense transgene carrying a small inverted repeat in the 5' untranslated region was introduced into tomato to test the role of dsRNA structure as an inducer of PTGS. Cosuppression of the endogenous acc gene occurred at a higher frequency in these plants than in those harboring only the p35S-ACC sense transgene without the inverted repeat (93).

Reports from several laboratories in the past few years have established that the loss in steady-state accumulation of the target mRNA is almost total if the designed transgene construct of the transgenic plant produces the nuclear transcript in the duplex conformation. Very recently it was reported that the expression of self-cRNA of plum pox virus under the control of rolC promoter caused degradation of transgenic viral RNA and as a result, the systemic disease resistance to challenge inoculum of plum pox virus occurred with a high frequency in transgenic Nicotiana benthamiana (170). This evidence points out that the production of dsRNA is required to initiate PTGS in plants. Based on this, plants carrying strongly transcribing transgenes in both the sense and antisense orientations are currently being produced that show strong PTGS features. These transgenic plants can silence endogene, invading viral RNA, or unwanted foreign genes in a sequence-specific and heritable manner.

Generally, the sense and antisense components of the above-mentioned transgenes are separated only by an intron to increase the efficacy of PTGS (43, 198). For example, Arabidopsis thaliana and Lycopersicon esculentum (tomato) plants were transformed with a transgene construct designed to generate self-complementary iaaM and ipt transcripts. iaaM and ipt are oncogenes of agrobacteria that are responsible for crown gall formation in infected plants. The transgenic lines retained susceptibility to Agrobacterium transformation but were highly refractory to tumorigenesis, providing functional resistance to crown gall disease by posttranscriptional degradation of the iaaM and ipt transcripts (72).

The phenomenon of RNAi first came into the limelight following the discovery by Fire et al. (78), who unequivocally demonstrated the biochemical nature of inducers in gene silencing by introducing purified dsRNA directly into the body of Caenorhabditis elegans. The investigators injected dsRNA corresponding to a 742-nucleotide segment of unc22 into either the gonad or body cavity region of an adult nematode. unc22 encodes an abundant but nonessential myofilament protein, and the decrease in unc22 activity is supposed to produce an increasingly severe twitching phenotype. The injected animal showed weak twitching, whereas the progeny individuals were strong twitchers. The investigators showed that similar loss-of-function individuals could also be generated with dsRNAs corresponding to four other nematode genes. The phenotypes produced by interference by various dsRNAs were extremely specific.

This experiment paved the way for easy production of null mutants, and the process of silencing a functional gene by exogenous application of dsRNA was termed RNA interference (RNAi). RNAi in C. elegans was also initiated simply by soaking the worms in a solution containing dsRNAs or by feeding the worms Escherichia coli organisms that expressed the dsRNAs (209). This is a very potent method, requiring only catalytic amounts of dsRNA per cell to silence gene expression. The silencing spread not only from the gut of the worm to the remainder of the body, but also through the germ line to several generations. These phenomena of RNAi have also been demonstrated to occur in Drosophila melanogaster and many other invertebrates and vertebrates.

Analyses of these virus-resistant plants revealed that the transgenes were highly transcribed in the nucleus, whereas the steady-state level of cytoplasmic mRNA was very low. Further analysis suggested that some of the transgenic mRNA molecules assumed the conformation of dsRNA, which triggered sequence-specific degradation of self and other homologous or cRNA sequences in the cytoplasm. Thus, in the virus-resistant lines, not only the transgene mRNAs but also the mRNA from the homologous endogenous gene and the invading viral RNA (with homology to the transgene) were degraded.

Another form of virus-induced gene silencing is the phenomenon of viral recovery itself. When Brassica napus was inoculated with cauliflower mosaic virus (a DNA virus), lesions at the site of virus entry were visible 5 to 7 days postinoculation. Symptoms of systemic infections were apparent by 10 to 14 days postinoculation. Symptoms were most prominent at 30 to 40 days postinoculation and declined thereafter (i.e., the plants recovered), with the newly emergent leaves remaining asymptomatic at 50 days postinoculation (5).

Figure 1 diagrammatically illustrates the systemic spread of RNAi in plants. Such recovery occurred by a PTGS-like mechanism because 19S and 35S RNAs encoded by the cauliflower mosaic virus were degraded while cauliflower mosaic virus DNA was still replicating in the nucleus. Induction of PTGS was visualized if the cauliflower mosaic virus infection and subsequent recovery were followed up in a transgenic B. napus expressing a p35S-GUS (ß-glucuronidase) transgene. At the site of inoculation, GUS silencing associated with local lesions was first observed 7 days postinoculation. GUS silencing eventually spread systemically, and the GUS activity of the entire plant was suppressed by 50 days postinoculation. In this particular example, cauliflower mosaic virus acted as the inducer of PTGS for the transgenes sharing homology with the virus within the transcribed region. However, the virus itself was also the target of the induced PTGS, since 19S and 35S RNAs were found degraded.

View larger version (35K): [in this window] [in a new window]   FIG. 1. Schematic illustration of systemic viral spread as well as RNAi and subsequent viral recovery in plants. Green and red indicate the presence and loss of GFP fluorescence, respectively, and orange denotes the presence of both colors. The red dots on leaves show viral lesions. The bold arrows indicate the stages of plant growth, and the leaves are numbered accordingly. An arrow with a thin line shows a newly emerged leaf recovered from viral attack.

Based on the principles of virus-induced gene silencing, vectors designed with the genome sequence of RNA viruses tobacco mosaic virus, potato virus X, and tobacco rattle virus are being widely used to knock down the expression of host genes. The characteristics of many plant genes were revealed by observing the loss-of-function-related phenotypic changes when the recombinant vectors incorporating the concerned host genes were introduced into plants (136). Of these vectors, the TRV-based are more promising because these are capable of inducing-meristematic gene silencing, which has not been possible to achieve with other RNA virus-based vectors. Meristematic gene silencing employing TGMV vectors has also been reported (173). Thus, virus-induced gene silencing-based techniques are extremely useful for studies related to functional genomics in plants.

IMPORTANT FEATURES OF RNA SILENCING

Top Previous Next References

 
Independently of one another, investigations on diverse organisms, labeled variously as PTGS in plants, RNAi in animals, quelling in fungi, and virus-induced gene silencing, have converged on a universal paradigm of gene regulation. The critical common components of the paradigm are that (i) the inducer is the dsRNA, (ii) the target RNA is degraded in a homology-dependent fashion, and, as we will see later, (iii) the degradative machinery requires a set of proteins which are similar in structure and function across most organisms. In most of these processes, certain invariant features are observed, including the formation of small interfering RNA (siRNA) and the organism-specific systemic transmission of silencing from its site of initiation.

The siRNAs resemble breakdown products of an E. coli RNase III-like digestion (13). In particular, each strand of siRNA has 5'-phosphate and 3'-hydroxyl termini and 2- to 3-nucleotide 3' overhangs. Interestingly, in vitro-synthesized siRNAs can, in turn, induce specific RNA degradation when added exogenously to Drosophila cell extracts (69). Specific inhibition of gene expression by these siRNAs has also been observed in many invertebrate and some vertebrate systems (67). Recently, Schwarz et al. (189) provided direct biochemical evidence that the siRNAs could act as guide RNAs for cognate mRNA degradation.

Similar evidence is also available for plant PTGS. The new tissues growing from a GUS-expressing scion grafted onto a GUS-silenced rootstock show progressive silencing of GUS expression (168). The silencing signal seems to spread by a nonmetabolic, gene-specific diffusible signal, which travels both between cells, through plasmadesmata, and long distances via the phloem (75). In the case of virus-induced gene silencing, the systemic character has also been revealed (185). To account for the gene specificity of a systemic signal, it has been proposed that the signal could be an RNA molecule (228). However, such processes are not universal, as these are not found in flies and mammals.

COMPONENTS OF GENE SILENCING

Top Previous Next References

 
Both genetic and biochemical approaches have been undertaken to understand the basis of silencing. Genetic screens were carried out in the fungus Neurospora crassa, the alga Chlamydomonas reinhardtii, the nematode Caenorhabditis elegans, and the plant A. thaliana to search for mutants defective in quelling, RNA interference, or PTGS. Analyses of these mutants led to the identification of host-encoded proteins involved in gene silencing and also revealed that a number of essential enzymes or factors are common to these processes. Some of the components identified serve as initiators, while others serve as effectors, amplifiers, and transmitters of the gene silencing process. In the years to come, many other components as well as their interrelations will be revealed. Here, we outline what is known so far.

The predicted C. elegans Dicer homologue, K12H4.8, was referred as DCR1 because it was demonstrated to be the functional ortholog of the Drosophila Dicer protein (173). The 8,165-bp DCR1 protein has a domain structure similar to that of the Drosophila Dicer protein. dcr1 mutants of C. elegans showed defects in RNAi of germ line-expressed genes but no effect on the RNAi response of somatic genes. These mutants were found to be sterile, suggesting the important role of this gene in germ line development apart from RNAi (119). CAF1 has been identified as a Dicer homologue in A. thaliana, but it is not involved in PTGS activity. The structure of CAF1 shows the presence of the four distinct domains that were identified in the Drosophila Dicer protein (17, 36, 108). Dicer homologues from many different sources have been identified; some recombinant Dicers have also been examined in vitro, and phylogenetic analysis of the known Dicer-like proteins indicates a common ancestry of these proteins (83).

Complete digestion by RNase III enzyme results in dsRNA fragments of 12 to 15 bp, half the size of siRNAs (235). The RNase III enzyme acts as a dimer and thus digests dsRNA with the help of two compound catalytic centers, whereas each monomer of the Dicer enzyme possesses two catalytic domains, with one of them deviating from the consensus catalytic sequences.

Recently, the crystal structure of the RNase III catalytic domain was solved, and this led to the model for generation of 23- to 28-mer diced siRNA products (20). In this model, the dimeric Dicer folds on the dsRNA substrate to produce four compound catalytic sites so that the two terminal sites having the maximum homology with the consensus RNase III catalytic sequence remain active, while the other two internal sites bearing partial homology lose functional significance. Thus, the diced products appear as the limit digests of the RNase III enzymes and are double the size of the normal 12- to 15-mer fragments. Such a model also predicts that certain changes in Dicer structure might modify the spacing between the two active terminal sites and thus generate siRNAs of variable sizes bearing species-specific imprints (98). Clearly, the crystal structure of Dicer is necessary to authenticate this model.

After partial purification of crude extracts through differential centrifugation and anion exchange chromatography, the nuclease cofractionated with a discrete 25-nucleotide RNA species. These results suggested that small RNAs were associated with sequence-specific nuclease and served as guides to target specific messages based upon sequence recognition. In another report, the multicomponent RNAi nuclease was purified to homogeneity as a ribonucleoprotein complex of 500 kDa (97). One of the protein components of this complex was identified as a member of the Argonaute family of proteins and was termed Argonaute2 (AGO2). AGO2 is homologous to RDE1, a protein required for dsRNA-mediated gene silencing in C. elegans. AGO2 is a 130-kDa protein containing polyglutamine residues, PAZ, and PIWI domains characteristic of members of the Argonaute gene family. The Argonaute family members have been linked both to the gene-silencing phenomenon and to the control of development in diverse species. The first link between Argonaute protein and RNAi was shown by isolation of rde1 mutants of C. elegans in a screen for RNAi-deficient mutants. Argonaute family members have been shown to be involved in RNAi in Neurospora crassa (QDE3) as well as in A. thaliana (AGO1) (75).

Recently, two independent groups identified additional components of the RISC complex. Hammond and group showed the presence of two RNA binding proteins, the Vasa intronic gene and dFMR proteins, in the RISC complex isolated from Drosophila flies (35). Of these, dFMR is a homologue of the human fragile X mental retardation protein. In a parallel study, Siomi and group also isolated a novel ribonucleoprotein complex from the Drosophila lysate that contained dFMRI, AGO2, a Drosophila homologue of p68 RNA helicase (Dmp68), and two ribosomal proteins, L5 and L11, along with 5S rRNA (106). Both of these groups showed not only the presence of these components in the RISC complex, but also interactions among these proteins in vitro. Other components of RISC have not been clearly established yet. Nevertheless, some of the proteins mentioned below could very well constitute the RISC complex.

In Neurospora crassa, three classes of quelling-defective mutants (qde1, qde2, and qde3) have been isolated (46). The qde3 gene has been cloned, and the sequence encodes a 1,955-amino-acid protein (48). The protein shows homology with several polypeptides belonging to the family of RecQ DNA helicases, which includes the human proteins for Bloom's syndrome and Werner's syndrome (238). In addition, QDE3 is believed to be involved in the activation step of gene silencing. The DNA helicase activity of QDE3 may function in the DNA-DNA interaction between introduced transgenes or with a putative endogenous gene required for gene-silencing activation by unwinding the double-stranded DNA. These interactions may induce changes in methylation or chromatin structure, producing an altered state that could result in aberrant RNA production. Thus, QDE3 protein may be more important for the transcriptional part of gene silencing, i.e., TGS.

When the RNAi sensitivity of several existing C. elegans mutants was examined, two mutant strains, mut2 and mut7, that had previously shown elevated levels of transposon mobilization also showed resistance to RNAi. Ketting et al. (116) identified a mutator gene, mut7, in C. elegans and characterized it at the molecular level. MUT7 was found to be homologous to proteins with 3'-5' exonuclease domains, such as Werner's syndrome protein and E. coli RNase D. It contained all the key catalytic residues for nuclease activity. A model was proposed in which MUT7 was speculated to play a role in repressing transposition by degrading the target mRNA with its exonuclease activity.

smg (suppressor of morphological effects on genitalia) mutants of C. elegans, defective in a process called nonsense-mediated decay, have been isolated (63). Seven smg genes which are involved in nonsense-mediated decay have been identified (29, 100). Since this process also involves RNA degradation, the function of these genes, if any, in the RNAi process was examined. Animals mutant for a subset of these genes, smg2, smg5, and smg6, were initially silenced by dsRNA but later showed rapid recovery from the effects of RNAi, unlike the wild-type worms, which remained silenced. Thus, these genes might affect the persistence of RNA interference. On the other hand, smg1, smg3, and smg4 mutant animals behaved like wild-type worms and did not recover from RNAi at all, indicating that these genes are not required for RNAi persistence. The smg5 and smg6 genes have not been cloned, but the smg2 gene shows homology to Saccharomyces cerevisiae upf1, which encodes an ATPase with RNA-binding and helicase activities.

The SMG proteins could unwind dsRNA to provide a template for amplification activity. In this way, the three SMG proteins might facilitate amplification of the silencing signal and cause persistence of the silenced state. Alternatively, SMG proteins could increase the number of dsRNA molecules by promoting endonucleolytic cleavage of existing dsRNA molecules, which has been observed in Drosophila flies. No SMG2 homologues have been identified in plants or fungi. However, a search of the A. thaliana genome sequence database revealed a number of candidates with either helicase and/or RNase domains.

In a recent report, Tijsterman et al. (208) showed that unlike sense oligomers, single-stranded oligomers of antisense polarity could induce gene silencing in C. elegans. The antisense RNA-induced gene silencing was explained by proposing that RNA synthesis was primed on the mRNA by antisense RNA, resulting in dsRNAs, which acted as substrates for Dicer-dependent degradation. Antisense RNAs showed a requirement for the mutator/RNAi genes mut7 and mut14 but acted independently of the RNAi genes rde1 and rde4 of C. elegans. The mut14 gene was cloned by genetic mapping and subsequent candidate gene approach. The MUT14 protein is a member of the family of putative RNA helicases that contain the signature DEAD box motif. These proteins are involved in diverse cellular functions. The helicase activity of MUT14 might thus act to permit de novo RNA synthesis on the target.

Dalmay et al. (54) identified an sde3 locus in A. thaliana plants which is required for the PTGS phenotype. They proposed that SDE3 protein might be involved in the production of dsRNA. SDE3 differs markedly from QDE3/MUT7 and has slight similarity to MUT6 in the helicase motif. Although it is highly similar to Upf1p and SMG2, it is unlikely that SDE3 is the functional homologue of Upf1p and SMG2 because it lacks important motifs (167). Notably, no SDE3 homologue was found in C. elegans, suggesting that SDE3-like proteins are regulators rather than essential cofactors of PTGS and are not used in C. elegans. This is further supported by the observation that sde3 mutant plants exhibit only partial loss of PTGS (55). The closest homologue of SDE3 as identified by BlastP was a mouse protein encoded by gb110 (91, 159). These SDE3 homologues have RNA helicase motifs that are quite distinct from those of the DEAD, DEAH, and Ski2p types of RNA helicase (134). It has been speculated that SDE3 and SMG2 are multifunctional RNA helicases involved in PTGS.

Genetic screens of Neurospora crassa (QDE1) (48) and A. thaliana (SDE1/SGS2) (54, 160) led to the identification of proteins which are similar to tomato RdRP (77, 187) and are required for quelling and PTGS, respectively. This testifies to the importance of RdRP in gene silencing. Cogoni et al. (45) cloned the qde1 gene from N. crassa. It encodes a 158-kDa protein which lacks the typical signal peptide or a transmembrane domain, indicating its intracellular location. Dalmay et al. (54) found that the 113-kDa Arabidopsis RdRP is encoded by sde1. It is a plant homologue of QDE1 in N. crassa and EGO1 in C. elegans, which are required for quelling and RNAi, respectively. The SDE1 protein is required for transgene silencing but not for virus-induced PTGS, suggesting that SDE1 might be required to produce dsRNA, the initiator of PTGS (54).

The dsRNA produced as an intermediate in virus replication by virus-encoded RdRP might induce PTGS itself, and thus SDE1 may not be required for virus-induced PTGS. Plants with the sde mutation grow and develop normally, excluding a role for sde in development or basic cellular function. Two PTGS-controlling genes, sgs2 and sgs3, were identified in A. thaliana by another group of workers (160). Later, it was found that sgs2 and sde1 are different descriptions of the same gene. On comparing the protein sequence of all the RdRPs, a conserved block was identified which seems to be crucial for RdRP function in PTGS and RNAi. sgs3 mutants have the same molecular and phenotypic characteristics as sgs2 mutants, but the SGS3 protein shows no significant similarity with any known putative proteins.

In C. elegans, EGO1, a protein required for RNAi, was found to be similar to tomato RdRP and the QDE1 protein of Neurospora crassa (197), as mentioned earlier. For a number of germ line-expressed genes, ego1 mutants were resistant to RNA interference. The ego1 transcript is found predominantly in the germ line. ego1 is thus yet another example of a gene encoding an RdRP-related protein with an essential developmental function. RdRP is speculated to play a role in the amplification of the dsRNA signal, allowing its spread throughout the organism (50, 77, 168, 221). The RdRP is also perhaps responsible for sustaining PTGS at the maintenance level even in the absence of the dsRNA that initiates the RNAi effect.

In spite of its omnipresence in different kinds of eukaryotic cells, RdRP homologues are not coded by either the Drosophila or human genome. Though the systemic characteristics of RNAi have not been revealed yet in either flies or humans, the amplification of siRNAs may be an essential step of RNAi even in these systems. Hence, it is important to know how these steps of RNAi are biochemically carried out in the absence of RdRP activity.

The SID1 polypeptide is predicted to be a 776-amino-acid membrane protein consisting of a signal peptide and 11 putative transmembrane domains. Based on the structure of SID1, it was suggested that it might act as a channel for the import or export of a systemic RNAi signal or might be necessary for endocytosis of the systemic RNAi signal, perhaps functioning as a receptor. No homologue of sid1 was detected in D. melanogaster, which may be consistent with the apparent lack of systemic RNAi in the organism (80, 174). However, the presence of SID homologues in humans and mice might hint at the systemic characteristics of RNAi in mammals.

Mello and colleagues (87) have proposed that rde1 and rde4 respond to dsRNA by producing a secondary extragenic agent that is used by the downstream genes rde2 and mut7 to target specific mRNAs for PTGS. According to this view, rde1 and rde4 act as initiators of RNAi whereas rde2 and mut7 are effectors. Various components of gene silencing have been listed in Table 2.

Top Previous Next References

The role of the small RNAs in RNAi was confirmed independently by Elbashir et al. (69), who showed that synthetic 21- to 23-nucleotide RNAs, when added to cell-free systems, were able to guide efficient degradation of homologous mRNAs. To assess directly if the siRNAs were the true intermediates in an RNAi reaction, Zamore et al. (240) fractionated both the unprocessed dsRNAs and processed dsRNAs from the Renilla luc dsRNA-treated cell-free Drosophila system and showed that only the fractions containing native siRNAs were able to bring about the cognate RNA degradation and their ability to degrade RNA was lost when these fractions were treated at 95°C for 5 min. These in vivo and in vitro studies thus provided the evidence that siRNAs are the true intermediates of the RNAi reaction.

Together with the experiments to identify siRNAs as the key molecules for the RNAi effect, several investigators carried out the logical search for polypeptides that could generate such molecules. Based on the binding and cleavage properties of E. coli RNase III enzymes, Bass (13) for the first time predicted the involvement RNase III-type endonucleases in the degradation of dsRNA to siRNAs. The RNase III enzyme makes staggered cuts in both strands of dsRNA, leaving a 3' overhang of 2 nucleotides. The first evidence for the involvement of RNase III enzyme in RNAi was provided by T. Tuschl’s group, who chemically analyzed the sequences of the 21- to 23-nucleotide RNAs generated by the processing of dsRNA in the Drosophila cell-free system. They showed the presence of 5'-phosphate, 3'-hydroxyl, and a 3' 2-nucleotide overhang and no modification of the sugar-phosphate backbone in the processed 21- to 23-nucleotide RNAs (69).

Two groups recently identified candidate enzymes involved in degradation by scanning the genomes of D. melanogaster and C. elegans for genes encoding proteins with RNase III signatures (17, 115). Bernstein et al. (17) showed that one of these identified genes, dicer in Drosophila, codes for the RNA processing enzyme that fragments dsRNA into 22-nucleotide fragments in vitro. An antiserum raised against Dicer could also immunoprecipitate a protein from the Drosophila extract or from S2 cell lysate, and these Dicer protein immunoprecipitates were able to produce RNAs of about 22 nucleotides from the dsRNA substrate. The direct correspondence in size of these RNAs with those generated from dsRNA by cell extract suggested a role of this protein in dsRNA degradation. The role of Dicer in RNAi was further confirmed by the fact that the introduction of Dicer dsRNA into Drosophila cells diminished the ability of the transfected cells to carry out RNAi in vitro. Similar experimental studies were carried out with C. elegans extract, and an ortholog of Dicer named DCR1 was identified.

A number of in vivo and in vitro experimental studies have shown that the production of 21- to 23-nucleotide RNAs from dsRNA requires ATP. The rate of 21- to 23-nucleotide RNA formation from corresponding dsRNAs has been shown to be six times slower in the Drosophila extract depleted for ATP by treatment with hexokinase and glucose (165). Bernstein et al. (17) and Ketting et al. (115) showed that the Dicer immunoprecipitates from D. melanogaster as well as S2 cell extracts and DCR1 immunoprecipitates from C. elegans extract required ATP for the production of 22-nucleotide RNAs (17, 115). Recently, Nykanen et al. (165) reduced ATP levels in Drosophila extract by 5,000-fold with a sensitive ATP depletion strategy and showed considerable reduction in the rate of siRNA production in the Drosophila cell extract. These experiments suggest that ATP controls the rate of siRNA formation. However, it is still unclear whether ATP is absolutely rate limiting for the production of siRNAs from dsRNA.

The RNase activity and dsRNA binding of 218-kDa recombinant human Dicer have also been examined in vitro (175). The enzyme generated siRNA products from dsRNA quite efficiently in the presence of Mg²⁺ and the absence of ATP. The RNase activity was sensitive to ionic interactions, whereas the dsRNA binding was quite effective in presence of high salt and did not require Mg²⁺ at all. The dsRNA binding domain is located at the C terminus of Dicer, which is separable from the helicase and PAZ motifs. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. In other systems, Dicer has also been found to complex with various other proteins (35, 106). Hence, it is possible that the Dicer RNase activity functions as a complex of proteins in vivo.

Lipardi et al. (135), while investigating the dsRNA-dependent degradation of target mRNA in a Drosophila embryo cell extract system, showed the generation of full-length cognate dsRNAs from labeled siRNAs at early time points. Both single-stranded RNAs (equivalent to target mRNA) and dsRNAs served as templates for copying by RdRP. New full-length dsRNAs were formed rapidly and cleaved. They also showed a strict requirement for the 3'-hydroxyl group and 5'-phosphate group on siRNAs for primer extension in the RdRP-mediated reaction (135).

Sijen et al. (193) further revealed the role of RdRP activity in RNAi. In an RNAi reaction, they observed the formation of new siRNA species corresponding to target mRNAs but different from trigger dsRNAs. They named these new siRNAs secondary siRNAs. With a primary trigger dsRNA specific for the lacZ region of the target mRNA that encoded a GFP-LacZ fusion protein, these authors demonstrated the degradation of a separate GFP mRNA target. This kind of RNAi induced by secondary siRNAs was named transitive RNAi. These authors demonstrated the requirement for the rrf1 gene, a C. elegans gene with sequence homology to RdRP, in the generation of secondary siRNAs and transitive RNAi (193).

Amplification of siRNAs might occur at various stages of the RNAi reaction and has been documented in plants, C. elegans, N. crassa, and Dictyostelium discoideum but not in flies and mammals (66). Though the RdRP activity is present in Drosophila embryo extract, as mentioned earlier, it is surprising that the fly genome does not code for RdRP. Additionally, numerous experiments also suggest that RdRP is not required for RNAi in D. melanogaster (98).

A few independent studies demonstrated the importance of the RISC complex in this part of RNAi reactions. The mRNA-cleaving RNA-protein complexes have also been referred to as siRNP (small interfering ribonucleoprotein particles). It is widely believed that this nuclease is probably different from Dicer, judging from the substrate requirements and the nature of the end products. Since the target cleavage site has been mapped to 11 or 12 nucleotides downstream of 5' end of the guide siRNA, a conformational rearrangement or a change in the composition of an siRNP ahead of the cleavage of target mRNA is postulated. Finally, the cleaved mRNAs are perhaps degraded by exoribonucleases (96).

A part of cleaved fragments of mRNA at the end of step 2 might also be converted to the duplex forms by the RdRP-like activity. These forms might have siRNA-like functions and eventually enter the pool of the amplification reaction. Thus, it is likely that amplification of the RNAi reaction takes place at both step 1 and step 2 of RNAi. In another model, it has been proposed that siRNAs do not act as primers for the RdRP-like enzymes, but instead assemble along the length of the target RNA and are then ligated together by an RNA ligase to generate cRNA. The cRNA and target RNA hybrid would then be diced by the DCR protein. All these models were summarized by Schwarz et al. (189). Most of the steps involved in the mechanism of RNAi have been illustrated schematically in Fig. 2.

View larger version (63K): [in this window] [in a new window]   FIG. 2. Two-step model for the mechanism of gene silencing induced by double-stranded RNA. In step I, dsRNA is cleaved by the Dicer enzyme to produce siRNAs. A putative kinase seems to maintain 5' phosphorylation at this step. The siRNAs have also been proposed to be responsible for nuclear DNA methylation (•) and systemic spread of silencing. Amplification might occur due to the presence of RdRP (). In step II, the siRNAs generated in step I bind to the nuclease complex (RISC). A helicase present in the complex might activate RISC by unwinding the siRNAs. The antisense component of siRNA in the RISC guides the complex towards the cognate mRNA (—), resulting in endonucleolytic cleavage () of the mRNA. RdDM, RNA-dependent DNA methylation.

Top Previous Next References

In plants, PTGS has been widely linked with RNA virus resistance mechanisms (219, 227). Plant RNA viruses are, in fact, both inducers and targets for PTGS and gene-silencing-defective mutants of plants show increased sensitivity to viral infections (160). The direct role of dsRNA in inhibiting viral infection has recently been demonstrated by Tenllado and Diaz-Ruiz (207). They showed that dsRNAs derived from viral replicase sequences could interfere with virus infection in a sequence-specific manner by directly delivering the dsRNAs to leaf cells either by mechanical coinoculation with the virus or via an Agrobacterium-mediated transient-expression approach. Successful interference with the infection of plants by representative viruses belonging to the tobamovirus, potyvirus, and alfamovirus genera has been demonstrated. These results support the view that a dsRNA intermediate in virus replication acts as an efficient initiator of PTGS in natural virus infections.

The clinching support for the notion that PTGS has evolved as an antiviral mechanism has come from reports that plant viruses encode proteins that are suppressors of PTGS (8, 25, 222). These suppressors have evolved to save the viral RNA genomes from the PTGS degradative machinery of host plants. Different types of viral suppressors have been identified through the use of a variety of silencing suppression assays. Suppressors HC-PRO, P1, and AC2 are one type (encoded by potyviruses, rice yellow mottle sobemovirus, and geminiviruses of subgroup III, respectively) that is able to activate GFP expression in all tissues of previously silenced GFP-expressing plants (222). HC-PRO reduces target mRNA degradation and is thus responsible for reduced accumulation of siRNAs (137, 145). The second type of suppressors include movement proteins, i.e., p25 of potato virus X, which are involved in curbing the systemic aspect of transgene-induced RNA silencing (220). The third type includes cytomegalovirus 2b protein, which is involved in systemic signal-mediated RNA silencing (60). The cytomegalovirus 2b protein is nucleus localized and also inhibits salicylic acid-mediated virus resistance (141). Other types of viral suppressors with undefined biochemical activities are also known (128). These findings not only provide the strongest support that PTGS functions as a natural, antiviral defense mechanism, but also offer valuable tools for dissecting the biochemical pathways of PTGS (128).

The PTGS degradative machinery can both detect and inactivate repetitive DNA sequences, suggesting it controls the expansion of repetitive elements, including endogenous genes (18). Although RNAi occurs in mammals and mammalian cell cultures, its role in animal virus protection is not clear. In mammals, dsRNA induces RNAi as well as interferon-mediated nonspecific RNA degradation and other nonspecific responses leading to blockage in protein synthesis and cell death (2). Thus, mammals seem to have evolved multiple mechanisms to detect and target dsRNA and to fight viruses. These various mechanisms may have different specificities or can function in distinct tissues or during development (210). A few other roles of RNAi in development and genome maintenance will be discussed in later sections.

MECHANISTIC DIFFERENCES AMONG THE BIOSYNTHETIC PATHWAYS OF siRNA

Top Previous Next References

 
Although the functional parallelism of gene silencing is quite apparent in plants and animals, a few unique attributes separate the pathways in these groups. For example, systemic spreading of the RNAi reaction from the site of initiation is known to occur in plants and worms (74, 79), but not in flies or mammals. The noteworthy distinct molecules that have been identified to cause differences at the pre-Dicer, Dicer, and post-Dicer stages of gene silencing pathways are mentioned below.

Homologues of SGS3 are unknown beyond the plant world. Even though HEN1 analogs are known in bacteria, yeasts, and animals, their roles in sense PTGS have not yet been identified. Likewise, SGS2 homologues are known in C. elegans, N. crassa, and Dictyostelium discoideum, but their roles at the pre-dicing stage have not been established yet in those systems. The equivalents of SGS2 in other animal systems are nonexistent both structurally and functionally (205). The role of worm AGO1 protein, i.e., RDE1, is also unique, as described earlier. AGO1 homologues are present in all eukaryotes, but they mostly function as a component (AGO2) of the animal RISC complex (32). The plant HEN1 protein is believed to be nuclear because of the presence of the nuclear localization signal at its N-terminal region (40). Since HEN1 is essential for plant PTGS (cosuppression), which is supposedly a cytoplasmic activity, the exploration of its subcellular distribution is of utmost importance. Boutet et al. (22) speculated that HEN1 could be a dsRNA stabilizing protein, and since many such proteins are known in the animal kingdom, it would be of interest to find animal analogs of the plant HEN1 protein. In fungus as well as the animal system, sense transgene-induced PTGS phenomena are known, but the machinery operative at the pre-dicing stage is still elusive.

The roles of plant SGS2, SGS3, AGO1, and HEN1 proteins may be limited at the stage of production of dsRNA from the transcript of sense transgenes, but no mechanism has been established regarding the presentation of the dsRNA to Dicer for the generation of siRNA. However, such a mechanism has been reported in C. elegans. The RDE4 and RDE1 (AGO1) proteins of C. elegans were reported as initiators of RNAi and speculated to have no mechanistic role in the downstream processes of RNAi (87, 203). Unlike the Arabidopsis AGO1 and HEN1 proteins, RDE4 and RDE1 proteins are required for RNAi even when the dsRNAs are produced intracellularly in transgenic worms (203), but the defects in RDE4 and RDE1 are of no consequence if exogenous siRNAs or short antisense RNAs drive the RNAi reaction (208). RDE4 binds tightly to dsRNA (during the RNAi reaction) by virtue of its two RNA-binding domains and is always found in a tight complex with RDE1 protein even in absence of the RNAi reaction. During RNAi, RDE4 is found in a complex with RDE1, Dicer (DCR1), and a conserved DEXH-box RNA helicase (DRH1/DRH2). Based on these observations and other genetic evidence, Tabara and coworkers postulated that RDE4 and RDE1 functioned together to detect and retain foreign dsRNA and present the dsRNA to DCR1 for processing into siRNAs (202). Analogs of the RDE4 and DRH proteins are found in many eukaryotes, including plants and humans, but their roles have not been defined yet.

Interestingly, both in vivo and in vitro data suggest that the end products of plant dicing activities are different from those of the animal Dicers. When uniformly³²P-labeled dsRNA was incubated with wheat germ extract, Zamore et al. (205) found that the dsRNA was chopped into siRNAs of two discrete size classes, one 21 nucleotides and the other 24 to 25 nucleotides long, whereas D. melanogaster and human Dicers generated only the 21-nucleotide siRNAs. Two similar size classes were also produced with cauliflower extract and were found independently in the set of 423 endogenous small RNAs cloned from A. thaliana. Thus, in plants, dicing activity leads to the generation of two distinct classes of siRNAs.

With specific synthetic siRNAs that supposedly bind tightly to and inhibit Dicer as competitors, Zamore et al. concluded that a different Dicer-like enzyme was responsible for the generation of each class of siRNA. These two distinct classes of siRNAs were reported first in vivo from transgenic plants bearing the silenced GFP sense transgenes (94). With an array of plant virus-encoded suppressors of gene silencing, Baulcombe et al. proposed that the 21-mer siRNAs controlled localized PTGS via mRNA degradation and the 24-mer siRNAs triggered systemic silencing and methylation of the homologous DNA. It remains to be seen whether this kind of dual dicing activity reflects any novel pathway intrinsic to plant RNAi. Interestingly, the longer (25-mer) siRNAs have also been detected in the natural RNAi biology of Trypanosoma brucei (61).

RdRP-dependent siRNA amplification and systemic spreading from the site of origin is another area where many system-specific variations have been noticed. RdRP homologues are not present in many organisms, so the mechanisms by which sense transgene-mediated PTGS are effected in those organisms remain a mystery (98). In other systems where RdRP is present, the biochemical steps and details of siRNA amplification may not necessarily be the same.

In C. elegans, RRF1 (a putative homologue of RdRP), along with other proteins, is required for RNAi even when the trigger dsRNA is expressed directly from the hairpin transgene in the nuclei of somatic tissues, whereas SGS2 (Arabidopsis RdRP) is dispensable for PTGS activity if induced directly by hairpin sense transgenes in A. thaliana. This suggests that the RdRP-mediated putative amplification steps of worms are different from those of plants (37). In plants, the SGS2-dependent spreading of silencing occurs from the region homologous to the trigger dsRNA into both the adjacent nonhomologous 5' and 3' regions of a target transgene (214). In contrast, spreading occurs only in the 5' region in worms and fungi, which is consistent with the primer-dependent 5'-3' copying activity of RdRP. Hence, in plants, the spread of silencing requires other activities (such as chromatin modification) in addition to that of RdRP (37).

In worms, tissue-specific variations of RdRP-dependent RNAi have also been reported, but not in plants or other systems. EGO1 is essential for RNAi in the germ line of C. elegans, whereas another RdRP homologue, RRF1, is required for silencing in soma (193, 197). Another intriguing observation is that the loss of function of RRF3 (third putative RdRP of worms) is responsible for the enhancement of sensitivity to RNAi in several tissues of C. elegans. Here, RRF3 acts as a negative regulator of RNAi, a fact difficult to reconcile with the postulated activity of RdRP (195). For systemic transmission of gene silencing, the membrane-bound SID1 protein of C. elegans and the plasmodesmatal connections of plants are implicated, but in both cases, the molecular nature of the moving signal has not been ascertained yet. An association between Dicer and the RdRP has been suspected in the case of Dictyostelium discoideum and C. elegans, but conclusive evidence is still lacking (37).

siRNA: SYNTHESIS, DELIVERY, AND GENE KNOCKDOWN

Top Previous Next References

 
The natural RNAi biology of eukaryotic cells offers a protection mechanism against foreign nucleic acids; however, only in the recent past has the exploitation of its mechanistic details sparked a revolution in the investigation of cellular gene functions. Transcriptional regulation with the dsRNA technology provides an easy means to identify cellular characteristics in response to both internal and external cues. However, the application of RNAi in higher eukaryotes, particularly mammalian cells, has been hampered by the presence of a number of dsRNA-triggered pathways that mediate nonspecific suppression of gene expression (152). These nonspecific responses to dsRNA are not triggered by dsRNAs shorter than 30 bp, including the siRNA duplexes. Moreover, studies in C. elegans and D. melanogaster have clearly demonstrated that synthetic siRNAs can produce effects similar to those of the long dsRNAs (69, 236). Based on these experimental analyses, siRNAs are now being optimized for systematic exploration of the function of genes in a variety of organisms.

Prior to the siRNA era, approaches such as gene targeting by homologous recombination, ribozymes, and antisense technologies were commonly