Although the word ‘revolution’ should not be used lightly in science, there is no other
way to describe the recent explosion in our awareness and understanding of RNA-mediated
gene silencing pathways. The central player in RNA-mediated gene silencing is a
double-stranded RNA (dsRNA) that is chopped into tiny RNAs by the enzyme Dicer. The tiny
RNAs associate with various silencing effector complexes and attach to homologous target
sequences (RNA or DNA) by basepairing. Depending on the protein composition of the effector
complex and the nature of the target sequence, the outcome can be either mRNA degradation,
translational repression, or genome modification, all of which silence gene expression
(Figure 1). Present in plants, animals, and many fungi, RNA-mediated gene silencing
pathways have essential roles in development, chromosome structure, and virus resistance.
Although the mechanistic details are still under investigation, RNA-mediated silencing has
already provided a powerful tool for studying gene function and spawned a fledgling
industry that aims to develop novel RNA-based therapeutics to treat human diseases
(Robinson 2004).
Many biologists first learned of RNA-mediated gene silencing in 1998 following the
discovery, in the nematode worm
Caenorhabditis elegans (Fire et al. 1998), of a process called
RNA interference (RNAi), in which dsRNA triggers sequence-specific mRNA degradation. The
roots of RNA-mediated silencing, however, can be traced back 15 years, when a handful of
botanical labs stumbled across strange cases of gene silencing in transgenic plants. To
highlight the many seminal contributions of plant scientists to the field, we offer here a
personal perspective on the origins and history of RNA-mediated gene silencing in
plants.
Early Silencing Phenomena
Starting in the late 1980s, biologists working with transgenic plants found themselves
confronted with a ‘bewildering array’ of unanticipated gene silencing phenomena
(Martienssen and Richards 1995). Most intriguing were cases in which silencing seemed to be
triggered by DNA or RNA sequence interactions, which could occur between two separate
transgenes that shared sequence homology or between a transgene and homologous plant gene.
Several early examples supplied the prototypes for two types of RNA-mediated gene silencing
that are recognized today. In one type, silencing results from a block in mRNA synthesis
(transcriptional gene silencing [TGS]); in the second type, silencing results from mRNA
degradation (posttranscriptional gene silencing [PTGS]) (Figure 1).
TGS was revealed when two different transgene complexes were introduced in sequential
steps into the tobacco genome. Each complex encoded different proteins, but contained
identical gene regulatory regions (promoters). Unexpectedly, the first transgene complex,
which was stably active on its own, often became silenced in the presence of the second
(Figure 2). The promoters of the silenced transgenes acquired DNA methylation, a genome
modification frequently associated with silencing. Silencing and methylation were reversed
when the transgene complexes segregated from each other in progeny, suggesting that
interactions between the common promoter regions triggered silencing and methylation
(Matzke et al. 1989; Park et al. 1996).
PTGS was discovered in two ways. One involved experiments to evaluate antisense
suppression, a promising approach at the time for selectively silencing plant gene
expression. In theory, antisense RNA encoded by a transgene should basepair to the
complementary mRNA of a plant gene, preventing its translation into protein. Although the
control ‘sense’ transgene RNAs are unable to basepair to mRNA and hence should not induce
silencing, they often inexplicably did (Smith et al. 1990). In another type of experiment,
efforts to enhance floral coloration in petunia by overexpressing a transgene encoding a
protein involved in pigment synthesis led paradoxically to partial or complete loss of
color (Figure 2). This resulted from coordinate silencing (‘cosuppression’) of both the
transgene and the homologous plant gene (Napoli et al. 1990; Van der Krol et al. 1990),
later shown to occur at the posttranscriptional level (De Carvalho et al. 1992; Van
Blokland et al. 1994) A related phenomenon, called quelling, was observed in the
filamentous fungus
Neurospora crassa (Romano and Macino 1992). Similarly to TGS,
PTGS was often associated with DNA methylation of transgene sequences (Ingelbrecht et al.
1994).
Two influential papers appeared in the early 1990s. One reported the discovery of
RNA-directed DNA methylation in transgenic tobacco plants (Wassenegger et al. 1994). This
was the earliest demonstration of RNA-induced modification of DNA, a process that we return
to below. A second study showed that plant RNA viruses could be both initiators and targets
of PTGS. Plants expressing a transgene encoding a truncated viral coat protein became
resistant to the corresponding virus, a state achieved by mutual degradation of viral RNA
and transgene mRNA (Lindbo et al. 1993). In addition to forging a link between RNA virus
resistance and PTGS, this study included a remarkably prescient model for PTGS that
featured an RNA-dependent RNA polymerase (RDR), small RNAs, and dsRNA, all of which were
later found to be important for the RNAi. PTGS was subsequently shown in 1997 to protect
plants naturally from virus infection (Covey et al. 1997; Ratcliff et al. 1997). Transgene
PTGS thus tapped into a preexisting natural mechanism for combating viruses.
To recap: by 1998—the year in which RNAi was reported—plant scientists had documented
sequence-specific RNA degradation (PTGS), sequence-specific DNA methylation that triggered
TGS, and RNA-directed DNA methylation. They had also proposed models for PTGS involving
dsRNA (Lindbo et al. 1993; Metzlaff et al. 1997), small RNAs, and RDR (Lindbo et al.
1993).
RNAi
RNAi was discovered in experiments designed to compare the silencing activity of
single-stranded RNAs (ssRNAs) (antisense or sense) with their dsRNA hybrid. While only
marginal silencing of a target gene was achieved after injecting worms with the individual
strands, injection of a sense–antisense mixture resulted in potent and specific silencing
(Fire et al. 1998). This unequivocally fingered dsRNA as the trigger of silencing. Shortly
thereafter, dsRNA was shown to provoke gene silencing in other organisms, including plants
(Waterhouse et al. 1998). Indeed, the relatedness of RNAi, PTGS, and quelling was confirmed
when genetic analyses in worms, plants, and
Neurospora identified common components in the respective
silencing pathways (Denli and Hannon 2003). This included the aforementioned RDR, which can
synthesize dsRNA from ssRNA templates (see Figure 1). PTGS is now accepted as the plant
equivalent of RNAi.
The discovery of RNAi established a requirement for dsRNA in silencing, but details of
the mechanism remained unclear. In 1999, plant scientists studying PTGS provided a crucial
clue when they detected small (approximately 25 nucleotide-long) RNAs corresponding to
silenced target genes in transgenic plants (Hamilton and Baulcombe 1999). They proposed
that the small RNAs provided the all-important specificity determinant for silencing.
Consistent with this, a rapid succession of studies in
Drosophila systems demonstrated that 21–23 nucleotide ‘short
interfering'RNAs (siRNAs), derived from cutting longer dsRNA, can guide mRNA cleavage
(Zamore et al. 2000; Elbashir et al. 2001); identified RISC (RNA-induced silencing
complex), a nuclease that associates with small RNAs and executes target mRNA cleavage
(Hammond et al. 2000); and identified Dicer, the enzyme that chops dsRNA into short RNAs
(Bernstein et al. 2001) (see Figure 1).
RNAi/PTGS was detected originally in experiments involving transgenes, injected RNAs, or
viruses. Did the RNAi machinery also generate small RNAs for host gene regulation?
Strikingly, the newly discovered siRNAs were the same size as several ‘small temporal’
RNAs, first identified in 1993 as important regulators of developmental timing in worms
(Lee et al. 1993; Reinhart et al. 2000). Everything came together in 2001 when heroic
cloning efforts unearthed dozens of natural small RNAs 21–25 nucleotides in length, first
from worms and flies and later from plants and mammals (Lai 2003; Bartel 2004). Similar to
siRNAs, the natural small RNAs, dubbed microRNAs (miRNAs), arise from Dicer processing of
dsRNA precursors and are incorporated into RISC (Denli and Hannon 2003). In many cases,
miRNAs effect silencing by basepairing to the 3′ ends of target mRNAs and repressing
translation (see Figure 1). miRNAs are now recognized as key regulators of plant and animal
development. Identifying their target genes and full range of action are areas of intense
research (Lai 2003; Bartel 2004).
Up until 2002, RNAi/PTGS and miRNAs were the most avidly studied aspects of RNA-mediated
gene silencing. The next major advance, however, abruptly turned attention back to
RNA-guided modifications of the genome. By 2001, plant scientists working on RNA-directed
DNA methylation and TGS had demonstrated a requirement for dsRNAs that are processed to
short RNAs, reinforcing a mechanistic link to PTGS (Mette et al. 2000; Sijen et al. 2001).
This established the principle of RNA-guided genome modifications, but the generality of
this process was uncertain because not all organisms methylate their DNA. Widespread
acceptance came with the discovery in 2002 of RNAimediated heterchromatin assembly in
fission yeast (Hall et al. 2002; Volpe et al. 2002). This silencing pathway uses short RNAs
produced by Dicer and other RNAi components to direct methylation of DNA-associated
proteins (histones), thus generating condensed, transcriptionally silent chromosome regions
(heterochromatin) (see Figure 1). Targets of this pathway include centromeres, which are
essential for normal chromosome segregation. The RNAi-dependent heterochromatin pathway has
been found in plants (Zilberman et al. 2003) and
Drosophila (Pal-Bhadra et al. 2004) and likely represents a
general means for creating condensed, silent chromosome domains.
More Lessons from Plants
Plant scientists can chalk up other ‘firsts’ in RNA-mediated gene silencing. Systemic
silencing, in which a silencing signal (short RNA or dsRNA) moves from cell to cell and
through the vascular system to induce silencing at distant sites, was initially detected in
plants in 1997 (Palauqui et al. 1997; Voinnet and Baulcombe 1997) and later in worms (Fire
et al. 1998), although not yet in
Drosophila or mammals. Viral proteins that suppress silencing by
disarming the PTGS-based antiviral defense mechanism were discovered by plant virologists
in 1998 (Anandalakshmi et al. 1998; Béclin et al. 1998; Brigneti et al. 1998; Kasschau and
Carrington 1998). One of these, the p19 protein of tombusviruses, acts as a size-selective
caliper to sequester short RNAs from the silencing machinery (Vargason et al. 2003). A
recent study suggests that animal viruses encode suppressors of RNA-mediated silencing (Li
et al. 2004).
Although RNA-mediated gene silencing pathways are evolutionarily conserved, there are
various elaborations in different organisms. For example, the plant
Arabidopsis has four Dicer-like (DCL) proteins, in contrast to
mammals and worms, whose genomes encode only one Dicer protein (Schauer et al. 2002). The
RDR family has also expanded in
Arabidopsis to include at least three active members. An
important goal has been to determine the functions of individual family members. Previous
studies in
Arabidopsis have shown that DCL1 is needed for processing miRNA
precursors important for plant development (Park et al. 2002; Reinhart et al. 2002), but
not for siRNAs active in RNAi (Finnegan et al. 2003). The paper by Xie et al. (2004) in
this issue of
PLoS Biology delineates distinct functions for DCL2, DCL3, and RDR2.
Nuclear-localized DCL3 acts with RDR2 to generate short RNAs that elicit DNA and histone
modifications; DCL2 produces short RNAs active in antiviral defense in the cytoplasm of
cells. This study illustrates nicely how RNA silencing components have diversified in
plants to carry out specialized functions.
By identifying small RNAs as agents of gene silencing that act at multiple levels
throughout the cell, molecular biologists have created a new paradigm for eukaryotic gene
regulation. Plant scientists have figured prominently in RNA-mediated silencing research.
Instrumental to their success was the early ability to produce large numbers of transgenic
plants, which displayed a rich variety of gene silencing phenomena that were amenable to
analysis. The agricultural biotechnology industry provided incentives to find ways to
stabilize transgene expression and use transgenic approaches to modulate plant gene
expression and to genetically engineer virus resistance. As exemplified by the petunia
cosuppression experiments, nonessential plant pigments provide conspicuous visual markers
that vividly reveal gene silencing. The history of gene silencing research shows once again
that plants offer outstanding experimental systems for elucidating general biological
principles.
