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Post-Transcriptional Gene Silencing in Plants

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Accumulating genetic and biochemical evidence suggests that antisense-mediated gene silencing, cosuppression, RNA interference and virus-induced gene silencing are all unique inputs into a common RNA silencing pathway triggered by double stranded RNA. This pathway, termed post-transcriptional gene silencing (PTGS) is characterized by accumulation of 21–25 nt small-interfering RNAs, sequence-specific degradation of target mRNAs, and methylation of target gene sequences. PTGS appears to be ancient and highly conserved, as several groups of homologous genes required for silencing in plants, animals, and fungi have been identified. Though biochemical dissection of PTGS is still in its infancy, several key activities have been identified, such as Dicer, the endonuclease responsible for synthesis of short-interfering RNAs, and RISC, the nucleoprotein complex which mediates mRNA degradation. Several lines of evidence suggest that PTGS plays a key role in viral defense in plants, but further study is required to investigate the intriguing possibility that PTGS can act as an endogenous gene control mechanism.

Introduction

The characterization of antisense RNA-mediated controls of plasmid replication and maintenance in prokaryotes prompted several studies of mRNA silencing in eukaryotic genes in the mid-1980s.1,2 Rothstein et al (1987) first demonstrated an “antisense effect” in intact plants by silencing an integrated nopaline synthase (nos) transgene through expression of antisense nos RNA in tobacco.3 Interestingly, this eukaryotic antisense silencing appeared to result from increased turnover of the targeted nos mRNA, in contrast to the inhibitory effects on translation4 or DNA synthesis5 commonly associated with antisense RNA in bacteria. A similar homology-dependent RNA degradation phenomenon, resulting from the over expression of an endogenous gene in transgenic plants, was discovered soon thereafter.6,7 These phenomena, termed antisense-mediated gene silencing and cosuppression, became powerful techniques for plant improvement and functional gene analysis, but relatively little was understood about their underlying mechanisms.8

The seminal discoveries that double stranded RNA (dsRNA) and plant viruses are potent initiators of gene silencing has recently invigorated basic research on silencing mechanisms.9,10 It has become apparent that the diverse threads of antisense-mediated gene silencing, cosuppression, dsRNA-mediated gene silencing (RNA interference), and virus-induced gene silencing converge with the synthesis of dsRNA.11 This dsRNA is digested to 21–25 nt small-interfering RNAs (siRNAs) which play an integral role in homology-dependent RNA turnover and methylation of homologous DNA coding sequences in plants.12 This dsRNA-triggered sequence-specific RNA degradation pathway has been termed post-transcriptional gene silencing (PTGS). Studies of PTGS mutants and cell-free extracts capable of in vitro PTGS have demonstrated that PTGS is genetically and mechanistically conserved among plants, animals, and fungi, suggesting an ancient origin prior to the divergence of these kingdoms.13 In plants, PTGS appears to be a significant component of viral defense and possesses functional and mechanistic similarities to other epigenetic gene control mechanisms, such as paramutation and transcriptional gene silencing.14 In the following review, we discuss the current understanding of natural and artificial RNA inducers of PTGS in plants, models of the RNA degradation mechanism, and potential roles of PTGS in plants.

Initiation of PTGS in Plants: Many Roads to dsRNA

Antisense Mediated Gene Silencing

Expression of antisense RNA from integrated transgenes was the first, and until recently the most widespread, method of initiating PTGS in plants.8 Antisense constructs, usually consisting of an inverted gene coding sequence driven by a strong constitutive promoter, are moderately efficient inducers of PTGS, with ˜5–20% of transformed individuals displaying a reduction in target mRNA accumulation.15,16 The extent of mRNA suppression is quite variable from individual to individual, ranging from no detectable effect to >99% reduction in steady state RNA levels.8 In addition, silencing is highly dependent upon antisense RNA:mRNA homology, with decreasing nucleotide sequence identity (to ˜70%) minimizing the extent of mRNA suppression.17 Nuclear run-on assays have demonstrated that antisense-silenced lines are generally not deficient in transcription of the target mRNA, indicating that silencing is a post-transcriptional effect.8

The presence of abundant double stranded siRNA in antisense-silenced plants strongly supports the long-standing hypothesis that homologous pairing of sense and antisense RNAs produces dsRNA in vivo, and that this dsRNA is the primary initiator of silencing.18,19 Although sense:antisense RNA duplex formation is likely the primary mechanism of dsRNA production in antisensed plants, Stam et al (2000) have reported that dsRNA can also arise from the read through transcription of antisense transgene constructs integrated as inverted repeats, resulting in production of self-complementary RNA molecules.20 Presumably the frequency and extent of PTGS in antisense RNA-expressing plant lines is dependent upon the efficiency of RNA:RNA hybridization, which may be affected at the individual gene level by secondary mRNA structure and at the individual transformant level by the abundance of antisense RNA.18

RNA Interference (RNAi)

In 1998, Fire et al demonstrated that dsRNA is a substantially more efficient inducer of PTGS in Caenorhabditis elegans than sense or antisense RNA alone.9 Microinjection of substoichiometric levels of dsRNA into the gut of C. elegans triggered highly-specific silencing of the targeted endogenous genes in 100% of tested individuals. Later studies determined that dsRNA-induced silencing occurs post-transcriptionally, through degradation of homologous mRNA.21 This highly efficient induction of PTGS by direct production of dsRNA, termed RNAi, was subsequently demonstrated in plants through expression of inverted-repeat transgenes or through simultaneous expression of sense and antisense transgenes.22 Several groups have now reported near-absolute suppression of various endogenous and transgene mRNA species in >90% of transformants by the expression of complementary gene sequences separated by a nonhomologous linker region or a spliceable intron.23,24 The stem-loop or hairpin RNAs resulting from transcription of these constructs must possess a dsRNA region of at least ˜100 nt to efficiently induce PTGS in plants, which likely explains why the limited secondary structure of endogenous mRNAs does not trigger silencing.16 Predictably, abundant siRNA is associated with RNAi in both plants and animals.13,16 The exceptional potency and efficiency of dsRNA as an inducer of PTGS was recently exemplified by the systematic functional analysis of C.elegans chromosomes I and III through feeding studies with dsRNA-expressing E. coli bacteria.25,26

Cosuppression

Cosuppression was first described in two similar studies of the chalcone synthase (chs) gene in Petunia.6,7 Attempts to increase gene expression by introduction of an additional copy of chs fused to a strong, constitutive promoter unexpectedly produced a significant number of transgenic plants displaying decreased abundance of both transgenic and endogenous chs mRNA, as manifested by wholly or partially white flower petals. This silencing caused by over expression of an endogenous gene was termed cosense-suppression, or cosuppression. Like antisense silencing and RNAi, cosuppression is a post-transcriptional, homology-dependent process associated with siRNA accumulation.15,19 As with antisense RNA, sense RNA transgene constructs are only moderately efficient inducers of PTGS (silencing in ˜5–20% of transformants).15,16 However, unlike antisense RNA and RNAi, the high-level expression of sense RNA provides no apparent mechanism for production of a dsRNA trigger of PTGS. Although read through transcription of inverted transgene inserts or spurious transcription of antisense RNAs from cryptic transcription start sites could potentially account for cosuppression in some cases,27 genetic evidence suggests that cosuppression is mechanistically separable from antisense silencing and RNAi.

Analysis of Arabidopsis thaliana mutant lines which are partially deficient in PTGS have revealed several components of a catalytic pathway that could produce dsRNA from overabundant sense RNA. Mutagenesis of plant lines displaying stable cosuppression of the GUS or GFP marker genes resulted in characterization of four unique genes that were required for silencing.2831 The sde1/sgs2 gene is highly homologous to plant RNA-dependent RNA polymerases (RdRP), and similar genes are required for silencing in Neurospora (qde1) and C. elegans (ego1, rrf1). Similarly, the putative helicase gene sde3,, has homologs in Chlamydomonas (mut6) and C. elegans (smg2) that are also required for silencing. A putative translation initiation factor (ago1) with homology to the silencing effectors qde2 (Neurospora) and rde1 (C. elegans) was also necessary for silencing, as was sgs3, a gene with no known homologs which may be unique to silencing in plants. The cross-kingdom conservation in sequence and function of these genes strongly indicates that they are ancient components of the gene silencing pathway. However, these genes appear to uniquely affect PTGS initiated by sense RNA, as recent studies have demonstrated that RNAi and virus-induced gene silencing (see below) is unaffected in sde1/sgs2, sde3, agoI, and sgs3 mutants.32 These results suggest that a unique catalytic mechanism, likely involving sde1/sgs2-mediated synthesis of antisense RNA from a sense mRNA template, mediates production of dsRNA in the cosuppression pathway. How specific sense mRNA species are targeted for this catalytic process is unknown, though various models have invoked the presence of an mRNA accumulation threshold or the production of ill-defined “aberrant RNA” molecules from highly expressed transgenes.15

In addition to the unique catalytic elements related to initiation of PTGS, cosuppressed plant lines also display a striking systemic spread of PTGS termed systemic acquired silencing.33 Voinnet and Baulcombe (1997) showed that transient ectopic expression of a sense GFP transgene in a single leaf of GFP-expressing Nicotiana benthamiana plants caused a local initiation of PTGS which gradually spread throughout the plant, resulting in almost complete silencing.34 The spread of PTGS mirrored the movement of cytoplasmic RNA viruses, with cell-to-cell transmission presumably through plasmodesmata and long-distance transmission through phloem. Similarly, several groups have demonstrated that cosuppression is graft-transmissible to nonsilenced plants.33,35,36 The systemic silencing signal is apparently stable and pervasive, as PTGS was transmitted from a GUS-cosuppressed rootstock to a GUS-expressing scion through 30 cm of wild-type (nonGUS-expressing) stem.33 Plants displaying antisense-mediated PTGS and RNAi appear incapable of systemic silencing (Escobar and Dandekar, unpublished results).36 This suggests that a signal required for systemic silencing is generated during the catalytic reactions specific to dsRNA production in the cosuppression pathway. The nature of this systemic silencing signal is unknown, but the sequence-specific nature of the signal suggests the existence of a nucleic acid component.

Virus-Induced Gene Silencing (VIGS)

Nepoviruses, caulimoviruses, and several other types of RNA virus induce an unusual pattern of symptom development, called recovery, on some host plants.10 Infected plants initially display severe localized symptom development and high viral load, but tissues which emerge subsequent to the initial infection are a symptomatic and show very low virus accumulation. These “recovered” tissues are resistant to subsequent infection by the inducing virus as well as other closely related viruses. Ratcliff et al (1997) demonstrated that viral recovery is mediated by a PTGS-like RNA degradation mechanism which is post-transcriptional and homology dependent.10,37 The finding that tobacco plants infected with potato virus X (PVX) accumulate PVX-homologous siRNA conclusively demonstrated that plant viruses can be potent initiators and targets of PTGS.12 Subsequently, several laboratories have developed recombinant viral vectors designed to initiate PTGS in plants.37,38 Silencing of transgenes and endogenous genes can be efficiently induced by the integration of as little as 23 nt of target gene sequence into these viral vectors.39

The utilization of PTGS-inducing recombinant viral vectors has allowed the dissection of several unique characteristics of VIGS. Like cosuppression, VIGS is systemic, as demonstrated by the spread of silencing from a single leaf infiltrated with a coat protein mutant (cell autonomous) recombinant PVX vector.40 The same study showed that the viral protein p25 is capable of suppressing the systemic silencing effect caused by either cosuppression or VIGS. Importantly, p25 had a differential effect on the local induction of silencing in the infiltrated leaf depending on whether the inducer of PTGS was sense RNA or virus. The high-efficiency induction of local silencing by over expression of sense mRNA was completely blocked by p25, but induction of PTGS in tissues expressing the viral vector was unaffected.40 In combination with mutant studies showing that VIGS can operate independent of several plant-encoded enzymes which are required for cosuppression (see above), these results suggest that viruses may produce dsRNA via two independent pathways.40 The direct production of dsRNA viral replication intermediates by the virus-encoded RdRP likely provides the sde1/sgs2-independent, p25-resistant inducer of PTGS. In addition, abundant single stranded viral genomic RNA and mRNA could independently trigger dsRNA synthesis through the plant-encoded, cosuppression-type pathway, thus leading to production of the systemic silencing signal.

A General Model for dsRNA Production in Plants

Homology dependent RNA degradation and siRNA, the key elements that unify antisense-mediated gene silencing, cosuppression, RNAi, and VIGS, are thought to act downstream of dsRNA production in the PTGS pathway.32,41 The evidence above suggests that the various initiators of PTGS in plants differ primarily in their mechanism(s) of dsRNA production.40,42 Cosuppression, and to some extent VIGS, appear to produce dsRNA from sense RNA via a catalytic mechanism requiring the plant genes sde1/sgs2, sde3, ago1, and sgs3. This “indirect” pathway is wholly inhibited by the p25 viral protein and generates an as yet uncharacterized systemic silencing signal. In contrast, antisense-mediated silencing, RNAi, and VIGS produce dsRNA through homologous pairing or through catalytic reactions independent of the plant-encoded genes described above. This “direct” pathway is p25-resistant and does not generate systemic silencing. A simplified binary model of dsRNA production in plants is presented in (Fig. 1).

Figure 1. Mechanisms of dsRNA production in plants.

Figure 1

Mechanisms of dsRNA production in plants. Analyses of PTGS mutants and viral suppressors of PTGS suggest that two primary pathways of dsRNA production exist in plants. In the direct pathway, dsRNA is generated through pairing of homologous RNA molecules (more...)

From dsRNA to Silencing Models of the RNA Degradation Pathway

The majority of significant insights regarding the RNA degradation mechanisms of PTGS have come from animal models, especially C. elegans and RNAi-competent cell-free Drosophilae extracts. Although PTGS appears to be highly conserved across kingdoms (see Table 1), it is possible that the specific mechanisms of PTGS in plants could differ significantly from the models described below and in (Fig. 2).

Table 1. Common elements of PTGS in animals, fungi, and plants.

Table 1

Common elements of PTGS in animals, fungi, and plants.

Figure 2. A model for the mode of action of PTGS.

Figure 2

A model for the mode of action of PTGS. Regions of dsRNA of >100 nt are digested by the RNAse III-type endonuclease Dicer, generating 21–25 nt siRNAs. siRNAs can be integrated into the nucleoprotein RNA-induced silencing complex (RISC), (more...)

Dicer and Primary siRNA

Cell-free extracts from Drosophilae embryos or S2 cells perform rapid, ATP-dependent cleavage of exogenously supplied dsRNA into discrete 21–22 nt siRNA molecules.43,44 Structurally, these siRNAs consist of a 19–20 nt double stranded region with 3' terminal single-stranded tails of 2 nt.45 This structure is characteristic of the products of RNAse III endonuclease digestion of dsRNA templates.45

Recently, Bernstein et al (2001) identified an RNAse III nuclease in Drosophilae which specifically cleaves dsRNA into ˜22 nt fragments in vitro.46 This ATP-dependent nuclease, called Dicer, possesses an amino terminal helicase domain, a PAZ domain, two RNAse III motifs, and a dsRNA binding motif. The function of glutamine-rich PAZ domains has not been experimentally determined, but this domain is shared with several members of the Argonaut gene family that are required for PTGS in various organisms.46 Dicer displays decreased activity on dsRNA substrates of less than 200 nt, which correlates with dsRNA size requirements for RNAi in Drosophilae. In addition, a 6–7 fold decrease in Dicer activity in vivo substantially reduces RNAi competence. These results suggest that Dicer is a key element in the synthesis of siRNA from dsRNA.46 Bioinformatic analyses have identified three unique genes encoding proteins with domain architecture identical to Drosophilae Dicer-1: C. elegans K12H4.8, Arabidopsis T25K16.4, and Arabidopsis carpel factory (caf) (Escobar and Dandekar, unpublished results).46 K12H4.8 was recently characterized as a functional ortholog of Dicer which is required for RNAi in C. elegans.47 The putative Arabidopsis Dicer orthologs T25K16.4 and CAF are 97% identical at the amino acid level, and previous developmental studies of caf mutants have suggested that this protein suppresses cell division in floral meristems.48 The functional importance of these proteins for PTGS in Arabidopsis is currently being investigated.

The RNA-Induced Silencing Complex (RISC)

Dicer is biochemically separable from the sequence-specific RNA degradation activity associated with PTGS, suggesting that the processes of siRNA production and mRNA turnover are performed by unique groups of enzymes.44 Hammond et al (2000) purified a ˜500 kD nucleoprotein complex, termed RISC, that mediates sequence-specific RNA degradation in vitro.44 The partial characterization of RISC has revealed a 22–25 nt RNA component (presumably siRNA) and a 130 kD protein component (Agronaute2), both of which are required for sequence-specific RNA degradation.44,49 Though the identity of the nuclease component has not yet been determined, the catalytic activity of RISC has been described in some detail. Exogenous application of synthetic 21–22 nt dsRNA molecules with structural characteristics identical to siRNA (see above) is sufficient to trigger silencing of homologous mRNA molecules in the in vitro Drosophilae system.45 By introducing a single synthetic siRNA species, Elbashir et al (2001) were able to precisely map the resultant cleavage site on homolgous mRNA molecules.45 Digestion of the mRNA occurred at a single site corresponding to the center of the siRNA molecule (11–12 nt from the 3' end of the siRNA). Presumably, the antisense strand of the siRNA component of RISC hybridizes with complementary mRNA, forming a short RNA duplex which is subsequently cleaved.45 This endonuclease reaction would generate substrates for nonspecific cellular exonucleases which could fully digest the mRNA.

Secondary siRNA

Several aspects of PTGS cannot be readily explained by a simple model which posits that the catalysis of dsRNA produces a population of primary siRNAs which act as the sole determinants of RISC specificity. For example, it is difficult to explain the massive RNA degradation response triggered in C. elegans by microinjection of tiny amounts of short (400–500 bp) dsRNA without the existence of some mechanism of dsRNA amplification.9 Further, several studies have shown that PTGS can target RNA sequences outside the original dsRNA inducer molecule. Sijen et al (2001) reported that a transcriptional fusion of the endogenous gene unc-22 and GFP (unc-22::GFP) could be silenced in C. elegans by microinjection of GFP dsRNA.50 This result was not unexpected, as the GFP sequences of the unc-22::GFP mRNA molecule should be degraded, destabilizing the entire transcript. However, the endogenous unc-22 gene (which possesses no homology to GFP) was also silenced, suggesting that target sequences for silencing were somehow expanded through some interaction with the unc-22::GFP transcript. This phenomenon was termed transitive silencing. Subsequent RNAse protection experiments showed accumulation of siRNAs homologous to unc-22, with a higher abundance of unc-22 sequences which lay closer to the unc-22/GFP junction in the fusion transcript.50 Mutant analyses demonstrated that the RNA-dependent RNA polymerase rrf-1 was required for production of these “secondary” siRNAs which could not have arisen directly from digestion of the introduced dsRNA molecule. In addition, rrf-1 mutants displayed a large decrease in total siRNA accumulation and were incapable of RNAi in somatic tissues.

Based upon this data, a model was proposed in which a relatively small population of primary siRNAs is derived from direct digestion of the introduced dsRNA molecules.50 These primary siRNAs can pair with homologous mRNA and directly or indirectly prime extension of an antisense RNA strand in the 5' to 3' direction by rrf-1. This catalytic activity would produce more dsRNA, ultimately generating a large population of secondary siRNAs, some of which would extend beyond the boundaries of the original dsRNA trigger. This amplification effect would appear to be required for PTGS, at least in the somatic tissues of C. elegans. However, the fact that transitive silencing has been described in Nicotiana benthamiana 51 suggests that a similar mechanism operates in plants, though it may play a less vital role in plants constitutively producing large amounts of dsRNA from integrated transgenes. It is also possible that systemic silencing in plants is caused by a related amplification effect in which a mobile signal molecule produced at the local PTGS initiation site primes de novo dsRNA synthesis from homologous mRNA templates in distant tissues.

DNA Methylation Induced by PTGS

In addition to a cytoplasmic, sequence-specific mRNA degradation activity, PTGS is also commonly associated with an increase in methylation of the target gene coding sequence in plants. Methylation of integrated transgenes has been correlated with antisense-silencing, cosuppression, RNAi, and VIGS, though endogenous genes may not be subject to this silencing-induced methylation.52,53 The fact that cytoplasmic RNA viral vectors can cause methylation of target genes in the nucleus suggests that methylation is mediated by a mobile, RNA-containing effecter, as has previously been hypothesized for viroid-induced methylation.54

The importance of methylation for initiation and maintenance of PTGS in plants remains unclear. Mutations in the Arabidopsis genes ddm1, a chromatin remodeling factor, or met1, a DNA methyltransferase, cause significant genome-wide reductions in DNA methylation. When ddm1-mutant Arabidopsis lines are crossed to lines displaying cosuppression of an integrated GUS transgene, a small fraction of the resultant progeny show a complete loss of silencing.55 Alternatively, a relatively high percentage of progeny from a cross between met1-mutant and GUS-silenced lines showed localized loss of PTGS in developing tissues at some time during development.55 Thus it would appear that methylation plays some role in the maintenance of PTGS in plants. However, this hypothesis is not supported by the results of Wang and Waterhouse (2000), who showed that chemical demethylation of DNA by 5-azacytidine treatment did not release PTGS of a GUS transgene in rice calli.52 Clearly, further studies will be necessary to determine the mechanism and functional importance of methylation induced by PTGS.

Roles of PTGS in Plants

Defense Against Viruses and Viroids

Several lines of evidence suggest that PTGS is an important defense mechanism against invasive nucleic acid parasites. DNA viruses, RNA viruses, and viroids are all initiators of PTGS in plants.38,56 Accordingly, PTGS mediates viral recovery and cross protection, whereby plants display a reduction in disease symptoms and develop de novo resistance to infection by viruses closely related to the primary inoculum.10 Further, loss of PTGS function in sde1/sgs2, sde3, ago1, and sgs3 mutants results in hypersensitivity to infection by certain viruses.32 However, the strongest evidence for an antiviral role of PTGS is the existence of viral proteins which suppress specific PTGS mechanisms in plants.

Although suppression of PTGS appears to be a widespread strategy among plant viruses,57 only three viral PTGS suppressors have been studied in detail: p25, 2b, and HC-Pro. As previously described, the potato virus X movement protein p25 presumably suppresses some aspect of the “indirect” dsRNA synthesis pathway (Fig. 1.), blocking systemic silencing and local silencing induced by cosuppression.40 Alternatively, the cucumoviral 2b protein has no discernible effect on local silencing, but prevents establishment of PTGS in tissues which emerge subsequent to viral infection.58 Thus, 2b most likely prevents synthesis or translocation of the systemic silencing signal and/or prevents the signal from initiating PTGS in distant tissues.59 Perhaps most interesting is the potyviral HC-Pro protein, which abolishes local silencing and siRNA accumulation, but does not block PTGS-associated methylation or systemic transmission of silencing to tissues which are not expressing HC-Pro.58 Yeast two-hybrid studies have shown that HC-Pro physically interacts with the calmodulin-related plant protein rgs-CaM.60 HC-Pro upregulates expression of rgs-CaM in vivo, and rgs-CaM alone has PTGS suppressor activity, so HC-Pro may operate primarily through manipulation of endogenous silencing-suppression pathways.60

Genome Stability

Indirect evidence suggests that PTGS may help to maintain genome integrity by suppressing the activity of transposable elements. Transposons in plant genomes are generally located in heterochromatic regions that are highly methylated and transcriptionally inactive. There is a correlation between decreased genome methylation and increased transposon activity in the ddm1 and met1 mutants of Arabidopsis, and these mutants also display defects in maintenance of PTGS, as described above.55 Similarly, the PTGS-deficient mutants mut7, rde2, and rde3 in C. elegans and mut6 in Chlamydomonas display transposon activation.61 In addition, the cloning and sequencing of siRNAs from tobacco has revealed a population of siRNAs homologous to integrated retrotransposon sequences.61 As both PTGS and transciptional gene silencing produce siRNAs and induce DNA methylation, it is likely that one or both of these pathways is involved in suppression of transposon activity in plants.

Regulation of Endogenous Gene Expression

It is tempting to speculate that an ancient and highly conserved pathway such as PTGS could play a broader role in the control of endogenous gene expression. At present there is no clear data to verify this hypothesis in plants, but several lines of evidence support the possibility. Mutants of some known and putative PTGS pathway components, notably ago1 and caf, display highly abnormal development, as do transgenic plants over expressing the endogenous PTGS suppressor rgs-CaM.30,48,60 Thus, it is possible that PTGS plays a role in development, but it is equally possible that ago1, caf, and rgs-CaM have separate developmental and PTGS-related activities. Naturally-occurring antisense RNA transcripts have been described for several endogenous genes in plants, but the functional importance of these antisense RNAs has not been determined.8 Currently, the only example of PTGS-like control of endogenous gene expression is the small temporal RNA (stRNA) system in C. elegans.47 The let-7 and lin-4 genes of C. elegans produce short, self-complementary mRNAs which form stem-loop structures in vivo. The duplex RNA sequence is digested into ˜21 nt fragments by Dicer, and these stRNAs suppress expression of various homologous mRNA targets which are involved in developmental timing. However, stRNA is thought to operate by hindering translation of mRNA rather than by the RNA degradation mechanism associated with siRNA and PTGS.47 Thus, several qualitatively different epigenetic control pathways may intersect with PTGS to coordinate endogenous gene regulation.

Conclusions

More has been learned about PTGS in the last four years than in the preceding fourteen years, dating back to the first demonstration of antisense-silencing in eukaryotes (Izant and Weintraub, 1984). In the near future, biochemical approaches using cell-free PTGS systems should allow further characterization of the nucleoprotein complexes mediating RNA degradation, DNA methylation, and secondary amplification of dsRNA, which should facilitate corresponding discoveries in plants. Still, many mysteries remain. Key questions of importance for students of PTGS in plants will be (1) What is the nature of the systemic silencing signal? (2) What is the functional relationship between methylation and PTGS? and (3) Does PTGS play a role in endogenous gene regulation? Further elucidation of the basic mechanisms of PTGS in the future should allow a continued refinement of gene silencing as a tool for functional genomics,26 plant biotechnology,62 and even human medicine.63

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