Download Biogenesis of trans-acting siRNAs, endogenous

Genes Genet. Syst. (2013) 88, p. 77–84
Biogenesis of trans-acting siRNAs, endogenous
secondary siRNAs in plants
Manabu Yoshikawa*
Division of Plant Sciences, National Institute of Agrobiological Sciences,
Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan
(Received 18 March 2013, accepted 3 April 2013)
Trans-acting small interfering RNAs (tasiRNAs) are plant-specific endogenous
siRNAs that control non-identical mRNAs via cleavage. The production of tasiRNAs
is triggered by cleavage of capped and polyadenylated primary TAS transcripts
(pri-TASs) by specific miRNAs. Following miRNA-directed cleavage, either 5′ or
3′ cleavage fragments are converted into double-stranded RNAs (dsRNAs) by RNADEPENDENT RNA POLYMERASE 6. The dsRNAs are processed to tasiRNAs by
DICER-LIKE 4 in a phasing manner. There are two forms of pri-TASs; One has
a single miRNA target site that is targeted by 22-nucleotide microRNAs, and the
other has two miR390 target sites. Secondary siRNAs that are important for the
amplification of RNA silencing are defined as siRNAs whose production is initiated
by the cleavage of primary small RNA-containing RNA-induced silencing
complexes. Thus, tasiRNA production is a model system of secondary siRNA production in plants. This review focuses on the production of tasiRNAs that are
endogenous secondary siRNAs.
Key words: ARGONAUTE, DICER-LIKE, microRNA, RNA-induced silencing
complex, trans-acting siRNA
INTRODUCTION
Phenomena that were previously described as RNA interference in nematodes, quelling in fungi, or co-suppression in
plants can be attributed to small RNA (sRNA)-mediated
sequence-specific gene regulation (Hannon, 2002). Two
protein families, Dicer and Argonaute (AGO), play central
roles in RNA silencing. Dicer proteins generate 20–25
nucleotide (nt) sRNA duplexes from double-stranded precursor RNAs. sRNAs produced by Dicer can be divided
into two types based on the precursor RNAs: microRNAs
(miRNAs) or small interfering RNAs (siRNAs). miRNAs
are excised from single-stranded RNAs (ssRNAs) that
form fold-back structures containing partially doublestranded regions. In contrast, siRNAs are processed
from perfectly complementary long double-stranded
RNAs (dsRNAs) that are formed between sense and antisense transcripts or are synthesized by RNA-dependent
RNA polymerases (RDRs). Plant Dicers, termed DICERLIKEs (DCLs) are phylogenetically grouped into the
DCL1, DCL2, DCL3 or DCL4 clades on the basis of four
Arabidopsis DCLs (Song et al., 2012; Mukherjee et al.,
2013). Each subclade DCL protein can generate sRNA
duplexes with distinct sizes from specific precursors. In
Edited by Hiroshi Iwasaki
* Corresponding author. E-mail: [email protected]
A. thaliana, DCL1 exclusively produces miRNAs from
long primary MIRNA transcripts via precursor miRNAs
(Kurihara and Watanabe, 2004). DCL2, DCL3 and
DCL4 generate 22 nt, 24 nt and 21 nt forms of siRNAs,
respectively (Xie et al., 2004; Bouche et al., 2006; Henderson
et al., 2006).
AGO family proteins associate with sRNAs and form
silencing effector complexes containing single-stranded
sRNA known as RNA-induced silencing complexes (RISCs).
RISCs can act on target RNAs with complementary
sequences and repress their targets post-transcriptionally
(by cleavage or translational inhibition) or transcriptionally (by DNA methylation). The A. thaliana genome
encodes 10 AGO genes that can be grouped into three
clades (Vaucheret, 2008; Borges et al., 2011). Molecular
analyses of these AGOs have revealed that AGOs function
in gene regulation redundantly, specifically or antagonistically depending on the targets (Bouche et al., 2006;
Katiyar-Agarwal et al., 2006; Baulcombe, 2007; Vaucheret,
2008). To achieve proper gene control, specific sRNA
recognition and loading into specific AGOs from a myriad
of sRNAs is critical. Although the forms of sRNA
duplexes are major determinants for sRNA loading into
AGOs in flies (Kawamata et al., 2009), the bases at the 5′
terminus of sRNAs dominate the specific sorting of individual sRNAs into specific AGOs in plants (Mi et al.,
2008; Takeda et al., 2008). Multiple AGO genes are
78
M. YOSHIKAWA
encoded in plants with large genomes (e.g., 18 in maize
and 19 in rice), and among them, some AGOs are finetuned to respond to specific conditions (Nonomura et al.,
2007; Wu et al., 2009; Qian et al., 2011; Singh et al.,
2011).
Several classes of functional endogenous siRNAs have
been defined in plants (Axtell, 2013). Heterochromatic
siRNAs derived from transposons or repetitive genomic
regions are 24 nt in length and they transcriptionally control the expression of target genes by DNA methylation,
a process called RNA-directed DNA methylation (RdDM)
(Qi et al., 2006). Cis-natural antisense siRNAs are produced from tail-to-tail 3′ overlapping transcripts between
neighboring genomic loci and regulate either transcript
(Borsani et al., 2005; Katiyar-Agarwal et al., 2006). Transacting siRNAs (tasiRNAs) that contain 21 nt are processed
from RDR-synthesized dsRNAs that are triggered by specific miRNA-guided target cleavages. TasiRNAs regulate
non-identical mRNAs via cleavage (Peragine et al., 2004;
Vazquez et al., 2004; Allen et al., 2005).
TasiRNAs were originally discovered in two independent studies on A. thaliana. In one study, genetic
screening of mutants whose phenotypes showed the
abnormality in the juvenile-to-adult transition was performed (Peragine et al., 2004). It has been shown that
these phenotypes result from upregulation of some auxin
response transcription factor genes regulated by tasiRNAs
generated from TAS3 (Table 1) (Adenot et al., 2006;
Fahlgren et al., 2006; Garcia et al., 2006; Hunter et al.,
2006). The other study analyzed transcripts of plants
with mutant RDR6 and SUPRESSOR OF GENE
SILENCING 3 (SGS3) genes that were defective in posttranscriptional gene silencing mediated by the sensetransgene (S-PTGS) (Vazquez et al., 2004). The production and function of tasiRNAs are unique in the following
ways: (i) their production is triggered by specific miRNA
cleavage of capped and polyadenylated primary TAS transcripts (pri-TASs), (ii) they are processed from either 5′ or
3′ miRNA-cleaved fragments in a 21 nt phasing manner
via conversion of dsRNAs (refer to Fig. 1 in Allen and
Howell, 2010 for the phased processing), and (iii) they
regulate non-identical mRNAs as well as miRNAs.
SiRNAs that arise from target RNAs beyond primary
sRNA target sites are called secondary siRNAs or transiTable 1.
Locus Trigger Trigger
names miRNAs AGOs
tive siRNAs (Baulcombe, 2007; Voinnet, 2008). Secondary siRNAs have been reported in nematodes and plants,
whose genomes encode multiple RDR genes (Sijen et al.,
2001; Vaistij et al., 2002). This indicates that the conversion of primary sRNA-containing RISC-cleaved targets
into dsRNAs by RDRs is an essential step in the production of secondary siRNAs. A current model of secondary
siRNA production is shown in Fig. 1. First, a primary
sRNA-programmed AGO-containing RISC cleaves a complementary target (step 1a in Fig. 1). In most cases,
RISC-cleaved RNAs are degraded (step 1b). However, a
subset of RISC-cleaved RNAs is involved in the production pathway of secondary siRNAs (step 2a). In plants,
22 nt forms of sRNAs and bulged forms of miRNA/
miRNA* duplexes lead RISC-cleaved RNAs into the pathway (Mlotshwa et al., 2008; Chen et al., 2010; Cuperus et
al., 2010). Their cleavage fragments are converted into
dsRNAs by RDRs (step 2a), after which they are processed into siRNAs by Dicers (step 3a) and their siRNAs
reinitiate further siRNA production (step 4a). Secondary
siRNAs are important for amplification and reinforcement of RNA silencing against RNAs that are harmful to
hosts, such as invading viruses (Ding, 2010). Because
the cleavage of pri-TASs by miRNAs is essential for
tasiRNA production, tasiRNAs belong to endogenous secondary siRNAs. Here, I review the steps in tasiRNA biogenesis in A. thaliana. Genes have been identified via
extensive genetic screenings for susceptibility to viruses
in Arabidopsis plants, the release of S-PTGS and developmental abnormality, and analyses have uncovered the
molecular functions of those genes in tasiRNA production.
CLEAVAGE OF PRIMARY TAS TRANSCRIPTS
BY miRNAs
Pri-TASs are transcribed from TAS loci in the genome
by DNA-dependent RNA polymerase II (RNA Pol II).
Thus, pri-TASs have a 5′ cap structure and 3′ polyadenylation similar to mRNAs. Eight TAS loci (TAS1a-c, TAS2,
TAS3a-c and TAS4) have been identified in A. thaliana
and each pri-TAS is approximately 1 kb in length (Table
1) (Peragine et al., 2004; Vazquez et al., 2004; Allen et al.,
2005; Yoshikawa et al., 2005; Rajagopalan et al., 2006;
Howell et al., 2007). Two distinct forms of pri-TASs have
TAS loci in Arabidopsis thaliana
Source of tasiRNAs
Target mRNAs
References
TAS1
miR173
AGO1
3′ cleavage fragment Pentatricopeptide repeat proteins Vazquez et al. (2004)
Unknown proteins
Peragine et al. (2004)
TAS2
miR173
AGO1
3′ cleavage fragment Pentatricopeptide repeat proteins Yoshikawa et al. (2005)
Allen et al. (2005)
TAS3
miR390
AGO7
5′ cleavage fragment ARF transcription factors
Allen et al. (2005)
TAS4
miR828
AGO1
3′ cleavage fragment MYB transcription factors
Rajagopalan et al. (2006)
Biogenesis of tasiRNAs in plants
79
Fig. 1. A model of secondary siRNA production. The initial step is the cleavage of targets by a primary sRNAcontaining RISC (1a). Most cleavage fragments are subsequently degraded by RNases (1b). RDR synthesizes complementary RNAs using parts of cleavage fragments as templates (2a). Dicer processes dsRNAs into secondary siRNAs
(3a). Emerged secondary siRNAs trigger further rounds of siRNA production, resulting in the amplification of RNA
silencing (4a).
been described using the nucleotide requirements of
miRNA target sites for tasiRNA biogenesis; whereas “onehit” forms of pri-TASs contain single miRNA target sites,
“two-hit” forms contain two miRNA target sites (Axtell et
al., 2006). Pri-TAS1a-c and pri-TAS2 contain the
miR173 target site and pri-TAS4 contains the miR828
target site. Both miR173 and miR828 are incorporated
into AGO1, which is the most extensively characterized
AGO protein in plants (Baumberger and Baulcombe,
2005; Mi et al., 2008; Takeda et al., 2008). In contrast,
pri-TAS3a-c, which are “two-hit” types, contain two target
sites for miR390. The polarities of the miRNA cleavage
fragments serving as sources of tasiRNAs also differ.
TasiRNAs of pri-TAS1a-c, pri-TAS2 and pri-TAS4 are
derived from the miRNA-cleaved 3′ fragments. However,
pri-TAS3a-c are cleaved at the 3′ miR390 target site but
not the 5′ miR390 target site, and tasiRNAs are derived
from the 5′ cleavage fragments. While TAS3 and
miR390 are well conserved in moss and higher plants, the
other TAS loci and their trigger miRNAs are unique in
Arabidopsis (Axtell et al., 2006).
pri-TAS1a-c, pri-TAS2 and pri-TAS4 The miRNA/
miRNA* duplexes are loaded into AGO1 via the molecular
chaperone HSP90 accompanied by the co-chaperone
SQUINT/CYCROPHILIN 40 (Iki et al., 2010, 2012; Earley
and Poethig, 2011). After incorporation of miRNA/
miRNA* duplexes into AGO1, miRNA*s are released and
miRNAs remain in AGO1, resulting in the formation of
RISCs. Although most plant miRNAs are 21 nt in
length, miR173 and miR828, which trigger tasiRNA production, are 22 nt. In fact, 21 nt forms of modified
miR173 and miR828 lose the ability to initiate tasiRNA
production in vivo (Chen et al., 2010; Cuperus et al.,
2010). On the other hand, bulged miRNA/miRNA*
duplexes of different sizes are competent enough to trigger secondary siRNA production. Contrary to the strict
requirements for miRNA length, target transcripts have
no necessity beyond the 22 nt miRNA target sites
(Montgomery et al., 2008b; Felippes and Weigel, 2009).
Experiments using an AGO1 defective in slicer activity
(the target cleavage activity) have demonstrated that the
RISC-directed cleavage event is essential for the tasiRNA
production (Carbonell et al., 2012).
pri-TAS3a-c The TAS3a locus has been identified as
the tasiRNA-generating locus initiated by miR390-guided
cleavage (Allen et al., 2005), and pri-TAS3 from the moss
80
M. YOSHIKAWA
Physcomitrella patens contains two miR390 target sites.
These dual miR390 complementary sites are well conserved in higher plants (Axtell et al., 2006). Axtell et al.
(2006) also confirmed that both miR390 target sites are
indispensable for tasiRNA production, and then proposed
a “two-hit trigger model.” Interestingly, Montgomery et al.
(2008a) showed that the 5′ target site is not cleaved by
miR390-AGO7 and cannot be swapped to other miRNA
target sites, while the 3′ target site is cleaved by miR390AGO7 and can be swapped to other AGO1-bound miRNA
target sites. This observation implies that the cleavage
event of the 3′ target site by a miRNA-guided RISC is
important regardless of RISCs containing AGO7 or
AGO1, but the recognition of miR390-binding AGO7 at
the 5′ target site is essential for tasiRNA production. An
additional difference between tasiRNA production from priTAS3 and other pri-TASs is that miR390 is 21 nt in length
and is exclusively associated with AGO7 (Montgomery et
al., 2008a). An experiment using the substitution of the
5′ nucleotide of miR390 suggested that specific loading of
miR390 into AGO7 is not affected by the 5′ terminal
nucleotides and the structure of the primary MIR390
transcript. The molecular mechanism by which specific
sorting of miR390 to AGO7 occurs remains unknown.
THO/TREX complex The accumulation of tasiRNAs
decreases in loss-of-function mutants of the THO/TREX
complex (Jauvion et al., 2010; Yelina et al., 2010). The
THO/TREX complex is composed of multiple subunits
that participate in the transportation of mRNAs from the
nucleus to the cytosol (Finnegan et al., 2003). In the
case of tasiRNA production, the molecular phenotypes of
tho mutants indicated a low abundance of tasiRNAs; overaccumulation of pri-TAS1 and pri-TAS2, but a low abundance of their miR173-cleaved fragments; an abundance
of miR173 to similar levels as that in wild-type. These
observations strongly suggest that the THO/TREX complex acts on pri-TASs at a step after transcription but
before miR173-AGO1 cleavage. However, the function of
this complex in tasiRNA production remains unclear.
Ribosome Zhang et al. (2012) examined the effects of
translation on tasiRNA production using modified GFP
mRNA constructs that contained an miR173-triggering
synthetic tasiRNA cassette before or after stop codons.
They found that a greater abundance of synthetic tasiRNAs
were produced from constructs whose cassettes were
located < 10 nt downstream of the stop codon, unlike
those in which the cassettes were located > 10 nt downstream of the stop codon. They discussed the possibility
that the interaction of ribosomes with miR173programmed RISCs affects the efficiency of tasiRNA production.
CONVERSION OF miRNA-CLEAVED TAS
FRAGMENTS TO dsRNAs
In general, miRNA-cleaved fragments are unstable due
to the lack of polyadenylation or cap structures. In
plants, the 3′ fragments generated by miRNA cleavage
are promptly degraded by XRN4, which is a 5′ to 3′ exonuclease, and other unidentified RNases (Souret et al.,
2004). Thus, the stabilization of the cleavage fragments
generated by miR390 or 22 nt miRNA-containing RISCs
is important for subsequent steps. The 5′ and 3′ miR173cleaved fragments accumulate more in the rdr6 mutant
than in wild-type, while the 3′ fragments disappear and
the 5′ fragments decrease in the sgs3 rdr6 double mutant
similar to the sgs3 mutant (Yoshikawa et al., 2005).
Given that SGS3 and RDR6 are genetically epistatic,
SGS3 may act on the miR173-cleaved fragments before
RDR6, and SGS3 may stabilize the miR173 cleavage
fragments. An extensive characterization of SGS3 protein expressed in Escherichia coli indicated that SGS3
can specifically bind to a dsRNA containing a 5′-overhang,
but cannot bind to an ssRNA or a dsRNA with a 3′ overhang (Fukunaga and Doudna, 2009). These results suggest that SGS3 acts on dsRNA with 5′ overhangs formed
during tasiRNA production, but contradicts the prediction
that SGS3 stabilizes the single-stranded miR173-cleaved
fragments. Using RISC prepared from extracts of evacuolated tobacco protoplasts (BYL), my group recently
found that SGS3 is recruited on miR173-containing
RISCs that bind to the TAS2 RNA and form a complex
containing RISC and the cleavage fragments (Yoshikawa
et al., 2013). The formation of the complex is disturbed
by a 1 nt deletion at the 3′ terminus of miR173 or a mismatch at the 3′ end of the miR173 target site on TAS2
RNA, resulting in the degradation of the 3′ fragments.
Furthermore, the depletion of endogenous tobacco SGS3
in BYL causes instability in the 3′ miR173-cleaved
fragments. These results indicate that the 22 nt form of
miR173 and SGS3 are important for the stabilization of
the miR173 cleavage fragments. The effects of basepairing at the 3′ end of miR173 on SGS3 imply that SGS3
binds to the dsRNA, resulting in the formation of a 5′
overhang between miR173 and its target. Although the
function of SGS3 remains unclear in terms of the production of tasiRNAs from pri-TAS3, SGS3 co-localizes with
AGO7 (Jouannet et al., 2012). Thus, SGS3 may also stabilize miR390-guided RISC-cleaved fragments in concert
with an RISC that contains miR390-AGO7.
RDR6 shows dsRNA synthesis activity that is complementary to an ssRNA in vitro (Curaba and Chen, 2008).
In fact, RDR6-dependent complementary RNAs to the 3′
fragment from the TAS2 transcript have been detected in
vivo (Yoshikawa et al., 2005). However, how RDR6 is
recruited to the miRNA-cleaved TAS RNA fragments
remains unknown. SGS3 and RDR6 co-localize to a spe-
Biogenesis of tasiRNAs in plants
cific compartment in the cytosol, designated the SGS3/
RDR6 body. SGS3/RDR6 bodies never overlap with Pbodies that contain AGO proteins and decapping enzymes
(Kumakura et al., 2009). However, some SGS3/RDR6
bodies are observed close to P-bodies. This might indicate the functional linkage between the SGS3/RDR6 body
and the P-body. In contrast, AGO7 co-localizes with the
SGS3/RDR6 body (Jouannet et al., 2012).
SDE5 Genetic screenings of PTGS release have recovered sde5 mutants (Hernandez-Pinzon et al., 2007; Jauvion
et al., 2010). TasiRNA accumulation is abolished in the
sde5 null mutant and pri-TAS1 and pri-TAS2 accumulate
to similar level in wild-type, rdr6 and sgs3. The cleavage
fragments from pri-TAS1 and pri-TAS2 accumulate more
in the sgs3 mutant and less in the rdr6 mutant (Jauvion
et al., 2010). These observations suggest that SDE5
functions in tasiRNA production after pri-TASs by
miRNA cleavage but before dsRNA formation by RDR6 in
concert with SGS3 and/or RDR6 after miRNA cleavage.
Although SDE5 is partially homologous to the human
mRNA export factor TAP, the biochemical activity of
SDE5 remains uncharacterized (Hernandez-Pinzon et al.,
2007).
PROCESSING OF dsRNAs INTO tasiRNAs
RDR6-synthesized dsRNAs are processed into 21 nt
tasiRNA duplexes with a 2 nt overhang at both 3′ termini
81
by a heterodimer composed of DCL4 and the dsRNAbinding protein DOUBLE-STRANDED RNA BINDING
PROTEIN 4 (DRB4) (Hiraguri et al., 2005; Adenot et al.,
2006; Nakazawa et al., 2007; Fukudome et al., 2011).
The processing of the dsRNAs by DCL4/DRB4 begins
from the miRNA cleavage site in a stepwise manner (Xie
et al., 2005). To regulate registered target mRNAs, the
21 nt processing that begins at the miRNA cleavage sites
is critical for the production of defined tasiRNAs from
each TAS locus (Allen et al., 2005; Yoshikawa et al.,
2005). DCL4 and DRB4 localize to the nucleus (Xie et
al., 2004; Hiraguri et al., 2005), whereas the SGS3/RDR6
body localizes to the cytosol (Kumakura et al., 2009;
Jouannet et al., 2012). These observations imply that
the dsRNAs synthesized in the SGS3/RDR6 body are
imported into the nucleus. Two recent studies found
that the tasiRNA pathway partially participates in crosstalk with the RdDM pathway that occurs in the nucleus
(Wu et al., 2012; Kanno et al., 2013). They found that
the downstream genomic region of the miR173 target site
on the TAS1c locus and the spanning genomic region
between the 5′ and 3′ miR390 target site on the TAS3a
locus are methylated in CG, CHG, and CHH contexts.
These methylation patterns are consistent with the
miRNA-cleaved fragments that are converted into dsRNAs
by RDR6. In fact, these methylations are affected by
loss-of function mutations in SGS3 and RDR6. Interestingly, these types of methylations are needed for
NUCLEAR RNA POLYMERASE E1 (the largest subunit
Fig. 2. A proposed model of tasiRNA production in Arabidopsis thaliana. Pri-TASs are transcribed by RNA pol II and the 22 nt
miRNA- or miR390-guided RISCs cleave pri-TASs in the cytosol. Either the 5′ or 3′ fragments are converted into dsRNAs by
RDR6. The dsRNAs are processed by the DCL4/DRB4 complex. The orange dashed line reveals a nuclear membrane. The black
lines and the black dashed lines reveal probable and putative steps, respectively.
82
M. YOSHIKAWA
of DNA-dependent RNA polymerase V), AGO4 and AGO6,
but not DCL2, DCL3, DCL4 and RNA-DEPENDENT
RNA POLYMERASE 2, implying that RDR6-synthesized
dsRNAs are processed by DCL1. DCL1-produced siRNAs
are bound to AGO4 and AGO6, and the RISCs containing
siRNA-bound AGO4 or AGO6 can induce methylation on
the corresponding TAS loci. Thus, because DCL1 localizes to the nucleus, these observations might support that
the hypothesis the processing of dsRNAs synthesized
from pri-TASs occurs in the nucleus.
PERSPECTIVE
TasiRNAs are associated with AGO1, AGO2 or AGO5
following the sRNA-sorting rule of the 5′ terminal bases
(Mi et al., 2008; Takeda et al., 2008). These AGO proteins are preferentially localized to the cytoplasm (Takeda
et al., 2008; Borges et al., 2011; Wang et al., 2011). If
tasiRNAs are loaded into AGO proteins in the cytoplasm,
the export of tasiRNA duplexes to the cytosol is necessary
(Fig. 2). Plant miRNA/miRNA* duplexes are exported
from the nucleus to the cytoplasm by HASTY (HST),
which is an ortholog to Exportin-5 in animals (Park et al.,
2005). Loss-of-function mutations in HST affect the
accumulation of most miRNAs, but do not affect the accumulation of tasiRNAs from TAS1 and TAS2 (Park et al.,
2005; Yoshikawa et al., 2005). This observation implies
that an unknown protein exports tasiRNA duplexes to the
cytoplasm. Alternatively, tasiRNA-containing RISCs
might act on target mRNAs in the nucleus. In fact, there
is some evidence that PTGS occurs in the plant nucleus
(Xie et al., 2003; Hoffer et al., 2011). Future studies are
required to elucidate this issue.
I thank Drs Tatsuo Kanno and Yoshihisa Ueno for helpful comments on the manuscript. M.Y. is supported in part by the
Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
REFERENCES
Adenot, X., Elmayan, T., Lauressergues, D., Boutet, S., Bouche,
N., Gasciolli, V., and Vaucheret, H. (2006) DRB4-dependent
TAS3 trans-acting siRNAs control leaf morphology through
AGO7. Curr. Biol. 16, 927–932.
Allen, E., and Howell, M. D. (2010) MiRNAs in the biogenesis of
trans-acting siRNAs in higher plants. Semin. Cell Dev.
Biol. 21, 798–804.
Allen, E., Xie, Z., Gustafson, A. M., and Carrington, J. C. (2005)
MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207–221.
Axtell, M. J. (2013) Classification and Comparison of Small
RNAs from Plants. Annu. Rev. Plant Biol. 64, 137–159.
Axtell, M. J., Jan, C., Rajagopalan, R., and Bartel, D. P. (2006)
A two-hit trigger for siRNA biogenesis in plants. Cell 127,
565–577.
Baulcombe, D. C. (2007) Amplified silencing. Science 315, 199–
200.
Baumberger, N., and Baulcombe, D. C. (2005) Arabidopsis
ARGONAUTE1 is an RNA Slicer that selectively recruits
microRNAs and short interfering RNAs. Proc. Natl. Acad.
Sci. USA 102, 11928–11933.
Borges, F., Pereira, P. A., Slotkin, R. K., Martienssen, R. A., and
Becker, J. D. (2011) MicroRNA activity in the Arabidopsis
male germline. J. Exp. Bot. 62, 1611–1620.
Borsani, O., Zhu, J., Verslues, P. E., Sunkar, R., and Zhu, J. K.
(2005) Endogenous siRNAs derived from a pair of natural
cis-antisense transcripts regulate salt tolerance in
Arabidopsis. Cell 123, 1279–1291.
Bouche, N., Lauressergues, D., Gasciolli, V., and Vaucheret, H.
(2006) An antagonistic function for Arabidopsis DCL2 in
development and a new function for DCL4 in generating
viral siRNAs. EMBO J. 25, 3347–3356.
Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Gilbert, K. B.,
Montgomery, T. A., Nguyen, T., Cuperus, J. T., and
Carrington, J. C. (2012) Functional analysis of three
Arabidopsis ARGONAUTES using slicer-defective mutants.
Plant Cell 24, 3613–3629.
Chen, H. M., Chen, L. T., Patel, K., Li, Y. H., Baulcombe, D. C.,
and Wu, S. H. (2010) 22-nucleotide RNAs trigger secondary
siRNA biogenesis in plants. Proc. Natl. Acad. Sci. USA
107, 15269–15274.
Cuperus, J. T., Carbonell, A., Fahlgren, N., Garcia-Ruiz, H.,
Burke, R. T., Takeda, A., Sullivan, C. M., Gilbert, S. D.,
Montgomery, T. A., and Carrington, J. C. (2010) Unique
functionality of 22-nt miRNAs in triggering RDR6-dependent
siRNA biogenesis from target transcripts in Arabidopsis.
Nat. Struct. Mol. Biol. 17, 997–1003.
Curaba, J., and Chen, X. (2008) Biochemical activities of
Arabidopsis RNA-dependent RNA polymerase 6. J. Biol.
Chem. 283, 3059–3066.
Ding, S. W. (2010) RNA-based antiviral immunity. Nat. Rev.
Immunol. 10, 632–644.
Earley, K. W., and Poethig, R. S. (2011) Binding of the cyclophilin 40 ortholog SQUINT to Hsp90 protein is required for
SQUINT function in Arabidopsis. J. Biol. Chem. 286,
38184–38189.
Fahlgren, N., Montgomery, T. A., Howell, M. D., Allen, E.,
Dvorak, S. K., Alexander, A. L., and Carrington, J. C. (2006)
Regulation of AUXIN RESPONSE FACTOR3 by TAS3 tasiRNA affects developmental timing and patterning in
Arabidopsis. Curr. Biol. 16, 939–944.
Felippes, F. F., and Weigel, D. (2009) Triggering the formation
of tasiRNAs in Arabidopsis thaliana: the role of microRNA
miR173. EMBO Rep. 10, 264–270.
Finnegan, E. J., Margis, R., and Waterhouse, P. M. (2003) Posttranscriptional gene silencing is not compromised in the
Arabidopsis CARPEL FACTORY (DICER-LIKE1) mutant, a
homolog of Dicer-1 from Drosophila. Curr. Biol. 13, 236–
240.
Fukudome, A., Kanaya, A., Egami, M., Nakazawa, Y., Hiraguri,
A., Moriyama, H., and Fukuhara, T. (2011) Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro
dsRNA-cleaving activity of Arabidopsis Dicer-like 4. RNA
17, 750–760.
Fukunaga, R., and Doudna, J. A. (2009) DsRNA with 5′ overhangs contributes to endogenous and antiviral RNA silencing pathways in plants. EMBO J. 28, 545–555.
Garcia, D., Collier, S. A., Byrne, M. E., and Martienssen, R. A.
(2006) Specification of leaf polarity in Arabidopsis via the
trans-acting siRNA pathway. Curr. Biol. 16, 933–938.
Hannon, G. J. (2002) RNA interference. Nature 418, 244–251.
Henderson, I. R., Zhang, X., Lu, C., Johnson, L., Meyers, B. C.,
Biogenesis of tasiRNAs in plants
Green, P. J., and Jacobsen, S. E. (2006) Dissecting
Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.
Nat. Genet. 38, 721–725.
Hernandez-Pinzon, I., Yelina, N. E., Schwach, F., Studholme, D.
J., Baulcombe, D. C., and Dalmay, T. (2007) SDE5, the
putative homologue of a human mRNA export factor, is
required for transgene silencing and accumulation of transacting endogenous siRNA. Plant J. 50, 140–148.
Hiraguri, A., Itoh, R., Kondo, N., Nomura, Y., Aizawa, D., Murai,
Y., Koiwa, H., Seki, M., Shinozaki, K., and Fukuhara, T.
(2005) Specific interactions between Dicer-like proteins and
HYL1/DRB-family dsRNA-binding proteins in Arabidopsis
thaliana. Plant Mol. Biol. 57, 173–188.
Hoffer, P., Ivashuta, S., Pontes, O., Vitins, A., Pikaard, C.,
Mroczka, A., Wagner, N., and Voelker, T. (2011) Posttranscriptional gene silencing in nuclei. Proc. Natl. Acad. Sci.
USA 108, 409–414.
Howell, M. D., Fahlgren, N., Chapman, E. J., Cumbie, J. S.,
Sullivan, C. M., Givan, S. A., Kasschau, K. D., and
Carrington, J. C. (2007) Genome-wide analysis of the RNADEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidopsis reveals dependency on miRNA- and
tasiRNA-directed targeting. Plant Cell 19, 926–942.
Hunter, C., Willmann, M. R., Wu, G., Yoshikawa, M., de la Luz
Gutierrez-Nava, M., and Poethig, R. S. (2006) Trans-acting
siRNA-mediated repression of ETTIN and ARF4 regulates
heteroblasty in Arabidopsis. Development 133, 2973–2981.
Iki, T., Yoshikawa, M., Nishikiori, M., Jaudal, M. C., MatsumotoYokoyama, E., Mitsuhara, I., Meshi, T., and Ishikawa, M.
(2010) In vitro assembly of plant RNA-induced silencing
complexes facilitated by molecular chaperone HSP90. Mol.
Cell 39, 282–291.
Iki, T., Yoshikawa, M., Meshi, T., and Ishikawa, M. (2012)
Cyclophilin 40 facilitates HSP90-mediated RISC assembly
in plants. EMBO J. 31, 267–278.
Jauvion, V., Elmayan, T., and Vaucheret, H. (2010) The conserved RNA trafficking proteins HPR1 and TEX1 are
involved in the production of endogenous and exogenous
small interfering RNA in Arabidopsis. Plant Cell 22,
2697–2709.
Jouannet, V., Moreno, A. B., Elmayan, T., Vaucheret, H., Crespi,
M. D., and Maizel, A. (2012) Cytoplasmic Arabidopsis AGO7
accumulates in membrane-associated siRNA bodies and is
required for ta-siRNA biogenesis. EMBO J. 31, 1704–1713.
Kanno, T., Yoshikawa, M., and Habu, Y. (2013) Locus-specific
requirements of DDR complexes for gene-body methylation
of TAS genes in Arabidopsis thaliana. Plant Mol. Biol.
Rep. in press.
Katiyar-Agarwal, S., Morgan, R., Dahlbeck, D., Borsani, O.,
Villegas, A., Jr., Zhu, J. K., Staskawicz, B. J., and Jin, H.
(2006) A pathogen-inducible endogenous siRNA in plant
immunity. Proc. Natl. Acad. Sci. USA 103, 18002–18007.
Kawamata, T., Seitz, H., and Tomari, Y. (2009) Structural
determinants of miRNAs for RISC loading and slicerindependent unwinding. Nat. Struct. Mol. Biol. 16, 953–
960.
Kumakura, N., Takeda, A., Fujioka, Y., Motose, H., Takano, R.,
and Watanabe, Y. (2009) SGS3 and RDR6 interact and colocalize in cytoplasmic SGS3/RDR6-bodies. FEBS Lett. 583,
1261–1266.
Kurihara, Y., and Watanabe, Y. (2004) Arabidopsis micro-RNA
biogenesis through Dicer-like 1 protein functions. Proc.
Natl. Acad. Sci. USA 101, 12753–12758.
Mi, S., Cai, T., Hu, Y., Chen, Y., Hodges, E., Ni, F., Wu, L., Li,
83
S., Zhou, H., Long, C., et al. (2008) Sorting of small RNAs
into Arabidopsis argonaute complexes is directed by the 5′
terminal nucleotide. Cell 133, 116–127.
Mlotshwa, S., Pruss, G. J., Peragine, A., Endres, M. W., Li, J.,
Chen, X., Poethig, R. S., Bowman, L. H., and Vance, V.
(2008) DICER-LIKE2 plays a primary role in transitive
silencing of transgenes in Arabidopsis. PLoS One 3, e1755.
Montgomery, T. A., Howell, M. D., Cuperus, J. T., Li, D., Hansen,
J. E., Alexander, A. L., Chapman, E. J., Fahlgren, N., Allen,
E., and Carrington, J. C. (2008a) Specificity of ARGONAUTE7miR390 interaction and dual functionality in TAS3 transacting siRNA formation. Cell 133, 128–141.
Montgomery, T. A., Yoo, S. J., Fahlgren, N., Gilbert, S. D., Howell,
M. D., Sullivan, C. M., Alexander, A., Nguyen, G., Allen, E.,
Ahn, J. H., et al. (2008b) AGO1-miR173 complex initiates
phased siRNA formation in plants. Proc. Natl. Acad. Sci.
USA 105, 20055–20062.
Mukherjee, K., Campos, H., and Kolaczkowski, B. (2013) Evolution of animal and plant dicers: early parallel duplications
and recurrent adaptation of antiviral RNA binding in
plants. Mol. Biol. Evol. 30, 627–641.
Nakazawa, Y., Hiraguri, A., Moriyama, H., and Fukuhara, T.
(2007) The dsRNA-binding protein DRB4 interacts with the
Dicer-like protein DCL4 in vivo and functions in the transacting siRNA pathway. Plant Mol. Biol. 63, 777–785.
Nonomura, K., Morohoshi, A., Nakano, M., Eiguchi, M., Miyao,
A., Hirochika, H., and Kurata, N. (2007) A germ cell-specific
gene of the ARGONAUTE family is essential for the progression of premeiotic mitosis and meiosis during sporogenesis in rice. Plant Cell 19, 2583–2594.
Park, M. Y., Wu, G., Gonzalez-Sulser, A., Vaucheret, H., and
Poethig, R. S. (2005) Nuclear processing and export of
microRNAs in Arabidopsis. Proc. Natl. Acad. Sci. USA
102, 3691–3696.
Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L., and Poethig,
R. S. (2004) SGS3 and SGS2/SDE1/RDR6 are required for
juvenile development and the production of trans-acting
siRNAs in Arabidopsis. Genes Dev. 18, 2368–2379.
Qi, Y., He, X., Wang, X. J., Kohany, O., Jurka, J., and Hannon,
G. J. (2006) Distinct catalytic and non-catalytic roles of
ARGONAUTE4 in RNA-directed DNA methylation.
Nature 443, 1008–1012.
Qian, Y., Cheng, Y., Cheng, X., Jiang, H., Zhu, S., and Cheng, B.
(2011) Identification and characterization of Dicer-like,
Argonaute and RNA-dependent RNA polymerase gene families in maize. Plant Cell Rep. 30, 1347–1363.
Rajagopalan, R., Vaucheret, H., Trejo, J., and Bartel, D. P.
(2006) A diverse and evolutionarily fluid set of microRNAs
in Arabidopsis thaliana. Genes Dev. 20, 3407–3425.
Sijen, T., Fleenor, J., Simmer, F., Thijssen, K. L., Parrish, S.,
Timmons, L., Plasterk, R. H., and Fire, A. (2001) On the role
of RNA amplification in dsRNA-triggered gene silencing.
Cell 107, 465–476.
Singh, M., Goel, S., Meeley, R. B., Dantec, C., Parrinello, H.,
Michaud, C., Leblanc, O., and Grimanelli, D. (2011) Production of viable gametes without meiosis in maize deficient for
an ARGONAUTE protein. Plant Cell 23, 443–458.
Song, X., Li, P., Zhai, J., Zhou, M., Ma, L., Liu, B., Jeong, D. H.,
Nakano, M., Cao, S., Liu, C., et al. (2012) Roles of DCL4 and
DCL3b in rice phased small RNA biogenesis. Plant J. 69,
462–474.
Souret, F. F., Kastenmayer, J. P., and Green, P. J. (2004)
AtXRN4 degrades mRNA in Arabidopsis and its substrates
include selected miRNA targets. Mol. Cell 15, 173–183.
Takeda, A., Iwasaki, S., Watanabe, T., Utsumi, M., and Watanabe,
84
M. YOSHIKAWA
Y. (2008) The mechanism selecting the guide strand from
small RNA duplexes is different among Argonaute proteins.
Plant Cell Physiol. 49, 493–500.
Vaistij, F. E., Jones, L., and Baulcombe, D. C. (2002) Spreading
of RNA targeting and DNA methylation in RNA silencing
requires transcription of the target gene and a putative
RNA-dependent RNA polymerase. Plant Cell 14, 857–867.
Vaucheret, H. (2008) Plant ARGONAUTES. Trends Plant Sci.
13, 350–358.
Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli,
V., Mallory, A. C., Hilbert, J. L., Bartel, D. P., and Crete, P.
(2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 16, 69–79.
Voinnet, O. (2008) Use, tolerance and avoidance of amplified
RNA silencing by plants. Trends Plant Sci. 13, 317–328.
Wang, W., Ye, R., Xin, Y., Fang, X., Li, C., Shi, H., Zhou, X., and
Qi, Y. (2011) An importin beta protein negatively regulates
microRNA activity in Arabidopsis. Plant Cell 23, 3565–
3576.
Wu, L., Zhang, Q., Zhou, H., Ni, F., Wu, X., and Qi, Y. (2009)
Rice microRNA effector complexes and targets. Plant Cell
21, 3421–3435.
Wu, L., Mao, L., and Qi, Y. (2012) Roles of DICER-LIKE and
ARGONAUTE proteins in TAS-derived small interfering
RNA-triggered DNA methylation. Plant Physiol. 160, 990–
999.
Xie, Z., Kasschau, K. D., and Carrington, J. C. (2003) Negative
feedback regulation of Dicer-Like1 in Arabidopsis by
microRNA-guided mRNA degradation. Curr. Biol. 13, 784–
789.
Xie, Z., Johansen, L. K., Gustafson, A. M., Kasschau, K. D., Lellis,
A. D., Zilberman, D., Jacobsen, S. E., and Carrington, J. C.
(2004) Genetic and functional diversification of small RNA
pathways in plants. PLoS Biol. 2, E104.
Xie, Z., Allen, E., Wilken, A., and Carrington, J. C. (2005)
DICER-LIKE 4 functions in trans-acting small interfering
RNA biogenesis and vegetative phase change in Arabidopsis
thaliana. Proc. Natl. Acad. Sci. USA 102, 12984–12989.
Yelina, N. E., Smith, L. M., Jones, A. M., Patel, K., Kelly, K. A.,
and Baulcombe, D. C. (2010) Putative Arabidopsis THO/
TREX mRNA export complex is involved in transgene and
endogenous siRNA biosynthesis. Proc. Natl. Acad. Sci.
USA 107, 13948–13953.
Yoshikawa, M., Peragine, A., Park, M. Y., and Poethig, R. S.
(2005) A pathway for the biogenesis of trans-acting siRNAs
in Arabidopsis. Genes Dev. 19, 2164–2175.
Yoshikawa, M., Iki, T., Tsutsui, Y., Miyashita, K., Poethig, R. S.,
Habu, Y., and Ishikawa, M. (2013) 3′ fragment of miR173programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3. Proc. Natl. Acad.
Sci. USA 110, 4117–4122.
Zhang, C., Ng, D. W., Lu, J., and Chen, Z. J. (2012) Roles of
target site location and sequence complementarity in transacting siRNA formation in Arabidopsis. Plant J. 69, 217–
226.