15-05-2014, 12:15 PM
RNA Silencing in Plants: Yesterday, Today, and Tomorrow
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INTRODUCTION
RNA silencing has become a major focus of molec-
ular biology and biomedical research around the
world. This is highlighted by a simple PubMed search
for ‘‘RNA silencing,’’ which retrieves almost 9,000
articles. Interest in gene silencing-related mechanisms
stemmed from the early 1990s, when this phenomenon
was first noted as a surprise observation by plant
scientists during the course of plant transformation
experiments, in which the introduction of a transgene
into the genome led to the silencing of both the
transgene and homologous endogenes. From these
initial studies, plant biologists have continued to
generate a wealth of information into not only gene
silencing mechanisms but also the complexity of these
biological pathways as well as revealing their multi-
level interactions with one another. The plant biology
community has also made significant advancements in
exploiting RNA silencing as a powerful tool for gene
function studies and crop improvements.
In this article, we (1) review the rich history of gene
silencing research and the knowledge it has generated
into our understanding of this fundamental mecha-
nism of gene regulation in plants; (2) describe exam-
ples of the current applications of RNA silencing in
crop plants; and (3) discuss improvements in RNA
silencing technology and its potential application in
plant science.
The miRNA Pathway
sRNAs are classed into two categories based on their
mode of biogenesis: siRNAs are processed from long,
perfectly double-stranded RNA, and miRNAs from
single-stranded RNA transcripts (transcribed from
MIR genes) that have the ability to fold back onto
themselves to produce imperfectly double-stranded
stem loop precursor structures. The first miRNA, lin-4,
was discovered in C. elegans in 1993 by Victor Ambros
(Lee et al., 1993), and today hundreds of miRNAs have
been identified in plants and animals, including sev-
eral hundred unique miRNAs in Arabidopsis alone
(Millar and Waterhouse, 2005). In Arabidopsis, the
primary-miRNA transcript is cleaved by DCL1 in the
nucleus with the help of the dsRBP, HYPONASTIC
LEAVES1 (HYL1), to produce the shorter precursor-
miRNA (pre-miRNA) dsRNA molecule. The first
DCL1-catalyzed cleavage step in the miRNA biogen-
esis pathway is made just below the miRNA duplex
region of the dsRNA stem loop (Lu and Fedoroff,
2000). The miRNA duplex is then released from the
pre-miRNA stem loop structure by the second cleav-
age step of the miRNA pathway, which is again
directed by the combined action of DCL1 and HYL1
(Vazquez et al., 2004). The two-nucleotide 3# over-
hangs of the liberated miRNA duplex are methylated
by the sRNA-specific methyltransferase HUA EN-
HANCER1 (HEN1). The duplexes of siRNAs are also
methylated by HEN1, a process that appears to be
plant specific and is assumed to protect all sRNA
species from polyuridylation and degradation (Chen
et al., 2002; Yu et al., 2005).
The rasiRNA and RdDM Pathway
Another RNA silencing-related pathway in Arabi-
dopsis that is regulated at the sRNA level is transcrip-
tional gene silencing (TGS), which is an epigenetic
mechanism resulting in the silencing of a transgene or
an endogenous gene through the inactivation of their
promoter sequences. DNA methylation is essential for
normal plant and animal development and is also a
hallmark of TGS (Mette et al., 2000). In fact, the ma-
jority of methylation in plants is associated with repeat
sequences, such as transposons, and methylation of
these sequences is thought to occur as a natural suppres-
sor to control their expression (Wassenegger, 2005). In
Arabidopsis, repeat sequences have been shown to be
an extremely rich source of a unique class of siRNAs,
termed rasiRNAs, which are of the 24-nucleotide size
class, and rasiRNAs have been suggested to direct
DNA methylation and hence to transcriptionally si-
lence repetitive DNA sequences in the plant genome
(Chan et al., 2005). Wassenegger and colleagues (1994)
were the first to demonstrate that homologous trans-
genes could be methylated following the replication of
introduced RNA viroid sequences, suggesting that an
RdDM mechanism was responsible. Jones et al. (1998)
went on to show that nuclear DNA could be methyl-
ated by introducing a homologous cytoplasmically
replicating RNA virus.
CONCLUSION
Plant biologists pioneering in homology-dependent
transgene silencing and pathogen-derived virus resis-
tance research in the early 1990s could not have
realized at the time that they had stumbled on one of
the most fundamental and conserved gene control
mechanisms in eukaryotic organisms. What they saw,
but could not fully understand at the time, including
cosuppression, RNA-mediated virus resistance, and
RdDM, represent the core aspects of what we know
today about the mechanisms and functions of RNA
silencing. The revelation of the dsRNA-induced mech-
anism in 1998 was a watershed event, leading to a vast
expansion of interest in researching the molecular
details and biological functions of RNA silencing in
all eukaryotes. This and subsequent discoveries of the
various related sRNA pathways revolutionized the
way we study gene regulation and developmental
control in plants and animals.