The pre-genomic era of ‘small RNA’ biology
Small RNAs were unknown for the major part of these ‘dark ages’, but a rich body of work starting from the 1980s, primarily conducted by those interested in transgene technologies and plant–virus interactions, established the concept of post-transcriptional gene silencing (PTGS), and culminated in the discovery of small RNAs in 1999. By the mid 1990s it was clear that a mechanism whereby RNA accumulation is suppressed in a sequence-specific manner, by homologous sequences from transgenes or RNA viruses, could not be explained by the paradigms of gene expression at the time. Models to explain the new phenomenon included an RNA intermediate: either an antisense RNA or even small RNAs. Moreover, work with transgenes and viruses also led to the concept of RNA-directed DNA methylation (RdDM), which we now recognize to play an essential role in genome stability. Therefore, the major concepts of RNA-directed RNA degradation and epigenetic modifications were established in the pre-genomic era, although almost none of the protein players in these processes were known at that time.
The desire to manipulate the expression of specific endogenous plant genes led to the discovery of PTGS. In the late 1980s, the phenomenon or technology known as ‘antisense RNA inhibition’, whereby an antisense RNA inhibits the expression/activities of homologous endogenous genes, first shown in animals, was established in plants. By transiently expressing sense and antisense constructs of the chloramphenicol acetyltransferase gene in protoplasts, Ecker and Davis (1986) showed that the antisense construct effectively suppressed the expression of the sense construct. In stably transformed plants, an antisense transgene was able to suppress the expression of a homologous sense transgene (Rothstein et al., 1987). The antisense technology was later found to be effective in the suppression of endogenous genes in stably transformed plants (van der Krol et al., 1988; Smith et al., 1988). Constitutive expression of an antisense chalcone synthase (CHS) gene led to reduced expression of the endogenous CHS gene, and altered floral pigmentation in both Petunia and tobacco (Nicotiana tabacum) (van der Krol et al., 1988). Similarly, expression of an antisense polygalacturonase gene in tomato (Solanum lycopersicum) suppressed the developmentally regulated endogenous gene (Smith et al., 1988). The levels of endogenous CHS and polygalacturonase mRNAs were drastically reduced.
Next, sense transgenes were unexpectedly found to also lead to the suppression of homologous endogenous genes (van der Krol et al., 1990; Napoli et al., 1990; Smith et al., 1990). With the aim of enhancing the colouration of Petunia petals, two groups sought to overexpress flavonoid biosynthesis genes, dihydroflavonol-4-reductase (DFR) or CHS, with either the strong cauliflower mosaic virus 35S promoter or the CHS endogenous promoter (van der Krol et al., 1990; Napoli et al., 1990). At a certain frequency, transgenic plants had reduced floral colouration, which could be attributed to reduced expression of both the endogenous genes and the transgenes (co-suppression). Similarly, constitutive expression of a truncated polygalacturonase gene in tomato resulted in a strong reduction in the levels of the endogenous polygalacturonase gene during fruit ripening (Smith et al., 1990). In the ensuing years, co-suppression was described for many transgene/endogenous gene pairs in a number of plant species (reviewed in Baulcombe, 1996). Moreover, nuclear run-on analyses revealed that this phenomenon occurred at the level of RNA degradation (de Carvalho et al., 1992; Ingelbrecht et al., 1994; Van Blokland et al., 1994; Elmayan and Vaucheret, 1996). The silenced transgenes and endogenous genes were being actively transcribed in the nucleus, but their RNAs failed to accumulate in the cytoplasm. Importantly, it was shown that a single copy 35S::uidA transgene with no sequence homology with any sequence in the tobacco genome underwent PTGS, ruling out models involving DNA–DNA interactions (Elmayan and Vaucheret, 1996).
Virus-induced gene silencing (VIGS)
A rich collection of research on plant RNA viruses was instrumental in revealing the role of PTGS as an anti-viral defense mechanism. Perhaps the earliest observation relating to VIGS as we know today was cross-protection, whereby inoculation of plant hosts with a mild viral strain protected the plants from subsequent infections by related, more severe viruses. These observations, as well as concepts of parasite-derived resistance in bacteria (Sanford and Johnston, 1985), led to a similar idea of pathogen-derived resistance whereby the introduction of pieces of the viral genome into plants may lead to resistance to homologous viruses. This idea was realized in 1986 when it was shown that the coat protein gene of tobacco mosaic virus (TMV), when introduced into tobacco, resulted in resistance against TMV (Abel et al., 1986). The subsequent years witnessed a flurry of similar transgenic efforts that effectively engineered resistance to many viruses in many plant species. However, the underlying molecular mechanisms were unknown. The first indication that at least some aspects of pathogen-derived resistance were RNA-based was from studies in which a non-translatable form of a viral coat protein gene was able to confer virus resistance when introduced into tobacco (van der Vlugt et al., 1992). Studies with pathogen-derived resistance with tobacco etch virus (TEV) finally revealed a link between viral resistance and PTGS (Lindbo et al., 1993). Transgenic tobacco lines expressing the full-length coat protein (CP) gene from TEV took time to develop anti-viral resistance, such that they were initially susceptible to TEV, but became resistant to TEV later in newly emerged leaves. The onset of viral resistance correlated temporally and spatially with the silencing of the CP gene, which was shown to be post-transcriptional by nuclear run-on experiments. The researchers predicted that a cytoplasmic RNA regulatory system of the host, which we now recognize as PTGS, was being triggered by the virus to lead to the simultaneous suppression of both viral and transgene RNAs. Subsequent studies with untranslatable versions of viral sequences corroborated the existence of such an RNA- and homology-based cytoplasmic regulatory system (Smith et al., 1994; Swaney et al., 1995). Since then, the concept that viruses initiate, and are targets of, PTGS was further established by findings that non-viral transgenes could silence viruses if the viruses were engineered to contain the transgenes, and that viruses were able to silence endogenous genes if they carried pieces of host genes (Kumagai et al., 1995; English et al., 1996; Angell and Baulcombe, 1997).
The mobile silencing signal
One intriguing aspect of PTGS in plants is its systemic nature. By grafting a non-silenced scion to a silenced rootstock containing the same transgene, it was shown that a silencing signal moved from the rootstock to the scion, leading to systemic silencing (Palauqui et al., 1997). The sequence-specific nature of the signal was demonstrated by combining a non-silenced scion with a rootstock containing a silenced transgene different from that in the scion. The systemic nature of PTGS was also revealed by an independent study where a GFP-expressing tobacco plant was infiltrated with Agrobacteria containing GFP within the T-DNA (Voinnet and Baulcombe, 1997). Systemic leaves that had not been infiltrated and from which no T-DNA could be detected showed silencing of GFP. Systemic silencing of GFP also occurred when GFP DNA was introduced into lower leaves by particle bombardment (Voinnet et al., 1998). By observing the patterns and timing of the initiation and spread of systemic silencing, it was concluded that the mobile signal travelled from cell to cell through the plasmodesmata, and systemically through the phloem. The sequence specificity of the silencing signal indicates that it is a nucleic acid, but its identity remained enigmatic at the time.
PTGS and RNA interference (RNAi)
In 1998, work in Caenorhabditis elegans revealed that injection of double-stranded RNA (dsRNA) into worms caused potent and specific repression of endogenous gene expression, a phenomenon termed RNAi (Fire et al., 1998). RNAi and PTGS were noted to share a number of common features, such as silencing at the level of mRNA degradation, sequence specificity and their systemic nature. Shortly after, dsRNA-containing transgenes were found to be the most effective trigger of PTGS in plants (Waterhouse et al., 1998; Chuang and Meyerowitz, 2000).
A report in 1989 (Matzke et al., 1989) first documented the phenomenon of homology-dependent de novo methylation. DNA methylation and inactivation of a transgene in tobacco occurred upon the introduction of a second T-DNA. Dependence on the second T-DNA for the methylation and inactivation of the first transgene was clearly established, and it was noted that the two T-DNAs shared regions of sequence identities, including identical promoters. A landmark study in 1994 demonstrated sequence-specific DNA methylation directed by RNA (Wassenegger et al., 1994). In this study, replication-competent and -incompetent versions of the potato spindle tuber viroid (PSTVd) cDNA were introduced into the tobacco genome. It was found that the transgene containing the replication-competent but not the replication-incompetent form was methylated. Infection of the transgenic plants containing the replication-incompetent transgene with the viroid induced methylation of the homologous transgene, but not other non-homologous sequences. This demonstrated that the replicating viroid RNAs specifically resulted in the methylation of the homologous DNA. Finally, it was shown that the silencing of a 35S promoter-driven transgene that conferred hygromycin resistance by another 35S promoter-containing transgene was accompanied by DNA methylation of the 35S promoter of the silenced transgene, and occurred at the level of transcription (Park et al., 1996). Thereby, homology-dependent promoter methylation was linked to transcriptional gene silencing (TGS).
Many studies also noted the methylation of coding sequences in PTGS (Ingelbrecht et al., 1994; Sijen et al., 1996; Van Houdt et al., 1997; Jones et al., 1998). The common theme in the TGS- and PTGS-associated methylation phenomena was that the methylation was largely restricted to regions of sequence homology between the trigger and the silenced loci. DNA–DNA and DNA–RNA interactions were proposed to explain the sequence specificity before dsRNA came to be known as the trigger of RNA silencing.
A major advance in the field of RNA silencing was the demonstration that TGS and PTGS are mechanistically similar, in that dsRNA was the most effective trigger in both processes. Two early studies found that when promoter sequences were arranged in inverted repeats to produce dsRNAs in vivo, homologous promoters elsewhere in the genome became methylated and transcriptionally silenced (Mette et al., 2000; Sijen et al., 2001).
Identification of small RNAs in plants
A major breakthrough in our understanding of TGS and PTGS in plants as well as RNAi in animals came in 1999, when Hamilton and Baulcombe (1999) reported small RNAs associated with PTGS. By fractionation of RNAs by gel electrophoresis followed by hybridization with probes corresponding to various transgenes undergoing PTGS by different triggers, they uncovered the presence of small RNAs matching to both strands of the transgenes in various plants (tomato, tobacco and Nicotiana benthamiana). The small RNAs were only present in plants undergoing PTGS of the transgenes. Small RNAs corresponding to viral sequences were also detected from plants infected with potato virus X. Consistent with the systemic nature of PTGS, small RNAs were detected in systemic tissues exhibiting silencing, triggered by Agroinfiltration of a single, basal leaf.
The discovery of small RNAs immediately shed light on the mechanism of PTGS or RNAi, which by 1998 was known to be triggered by dsRNA (Fire et al., 1998). The fact that the small RNAs associated with PTGS were of both sense and antisense orientations immediately suggested that they are processed from long dsRNA triggers by an endonuclease. It was also natural to speculate that the small RNAs served as the sequence determinants in guiding target RNA degradation. Indeed, biochemical studies performed with Drosophila in vitro RNAi systems uncovered RNA-guided target mRNA degradation at 21–23-nt intervals (Hammond et al., 2000; Zamore et al., 2000). These and other studies were to eventually establish a framework of RNA silencing, thereby revealing a new paradigm of gene regulation.
Although the persistent pursuit by a small number of research groups to unravel the mystery behind homology-dependent gene silencing eventually established the concept of RNA-mediated gene silencing, research conducted by developmental biologists unknowingly laid the foundation for the molecular framework of gene silencing by a class of endogenous small RNAs: microRNAs (miRNAs). Many plant miRNAs target transcription factor mRNAs, and play essential roles in plant development. Consequently, genes that participate in miRNA biogenesis or mediate miRNA functions are expected to mutate to pleiotropic developmental defects. A number of genes that play essential roles in miRNA biogenesis or function as we recognize today were identified in the pre-genomic era for various developmental defects that the corresponding mutants displayed, although the molecular functions of the genes were unknown. Mutants in DCL1, the major miRNA-generating Dicer, were isolated in at least three genetic screens aimed at identifying genes acting in different developmental processes. Severe dcl1 alleles were isolated as embryo-lethal mutants, and were found to be defective in embryo and suspensor development (Schwartz et al., 1994; McElver et al., 2001). Less severe dcl1 alleles were found to have short integuments (Robinson-Beers et al., 1992; Ray et al., 1996), or enhance other floral mutants to lead to the over-proliferation of the floral meristem (Jacobsen et al., 1999). A mutation in the miRNA biogenesis gene SERRATE (SE) was found to cause abnormal embryogenesis and accelerated phase changes (Clarke et al., 1999). A mutant in another miRNA biogenesis gene, HYPONASTIC LEAVES 1 (HYL1), exhibited leaf morphological defects and altered sensitivity to various plant hormones (Lu and Fedoroff, 2000). The developmental functions of two argonaute genes, AGO1 and AGO10 (also known as ZWILLE or PINHEAD), were also studied. ago1 mutants displayed severe defects in overall plant architecture, including radialized leaves, abnormal and sterile flowers, and lack of inflorescence branching (Bohmert et al., 1998). The defects of ago10 mutants were more restricted and less severe (Moussian et al., 1998; Lynn et al., 1999). These mutants failed to faithfully maintain the stem cells of the shoot apical meristem, and exhibited mild phenotypes in floral organs and ovules. When first cloned, the plant proteins were found to be similar to a rabbit protein eIF2C, which was so named for its ability to stimulate translation initiation in vitro (Bohmert et al., 1998; Moussian et al., 1998; Zou et al., 1998; Lynn et al., 1999). Since then, eIF2C proteins, now known as argonaute proteins, were found to be broadly conserved among diverse lineages of life, and their in vivo silencing functions were first revealed through genetic analysis in Drosophila and C. elegans (Schmidt et al., 1999; Tabara et al., 1999). Another gene, HUA ENHANCER 1 (HEN1), was also identified in a genetic screen for genes acting in flower development (Chen et al., 2002), and was later shown to be an integral player in various small RNA pathways.