millennium, multiple small RNA pathways were discovered in
plants that have related biochemical machineries but distinct
endogenous triggers and biological functions [3–12].
microRNA (miRNA) and trans-acting siRNA (tasiRNA) pathways are critical for plant development, affecting developmental
timing, organ identity, and other key traits [13–15]. miRNA biogenesis initiates when RNA polymerase II (Pol II) transcribes
MIRNA genes into primary miRNA transcripts (pri-miRNAs),
which fold into stem-loop hairpin RNAs (Fig. 1a, left). In terrestrial
plants these primary miRNAs and subsequent pre-miRNAs are
processed by Dicer-like 1 (DCL1) to produce ~21 nt miRNAs.
Different miRNAs can then guide Argonaute 1 (AGO1) to either
target mRNAs for translational inhibition or guide cleavage of
mRNAs [16–18]. A separate pathway produces tasiRNAs from
Pol II-transcribed TAS genes (Fig. 1a, right). TAS transcripts are
targeted by miRNA-guided binding/cleavage events (not shown)
that trigger the production of dsRNA by RNA-dependent RNA
polymerase 6 (RDR6). Dicer-like 4 (DCL4) processes these
dsRNAs into 21 nt siRNAs, which can target AGO1 to complementary mRNAs and thereby regulate development [7, 19, 20].
Transposable elements (TEs) constitute a large percentage of
many plant genomes, sometimes exceeding 80% in a given crop
species, such as maize [21]. In terrestrial plants, a class of ~24 nt
siRNAs silences TEs via RNA-directed DNA methylation
[22]. This process initiates with the recruitment of RNA polymerase IV (Pol IV) to a locus (Fig. 1b). Pol IV is an enzyme related to
Pol II but with distinct protein subunits, domains, and motifs
specialized for its function in TE silencing [6, 23–26]. Pol IV
transcribes chromosomal TEs and DNA repeats, channeling short
non-coding RNAs to RNA-dependent RNA polymerase 2 (RDR2)
[27, 28]. RDR2 then produces a second RNA strand, releasing
~30 bp dsRNAs that are processed by Dicer-like 3 (DCL3) [28–
30]. Resulting 24 nt siRNAs guide AGO4-clade proteins to sites of
RNA polymerase V transcription, leading to DNA methylation by
Domains Rearranged Methyltransferase 2 [31]. Pol IV-dependent
DNA methylation is accompanied by repressive histone modifications that silence TEs and protect genome integrity [23, 32–34].
TEs can be activated in response to environmental stresses
[35, 36]. For example, the Arabidopsis thaliana LTR retrotransposon ONSEN is activated under acute heat stress conditions [33, 37,
38]. After many plant generations exposed to selective pressure,
retrotransposition events may sometimes enable an adaptive stress
response. However, they more frequently cause deleterious loss-offunction alleles or ectopic expression of genes. Detecting and
annotating TEs is therefore an important, recurring task in functional and comparative genomics [21, 39, 40]. Because the majority of siRNA clusters overlap with TEs in plant genomes, small RNA
sequencing (small RNA-seq) can assist in the TE annotation
388
Marcel Bo ¨ hrer et al.
plants that have related biochemical machineries but distinct
endogenous triggers and biological functions [3–12].
microRNA (miRNA) and trans-acting siRNA (tasiRNA) pathways are critical for plant development, affecting developmental
timing, organ identity, and other key traits [13–15]. miRNA biogenesis initiates when RNA polymerase II (Pol II) transcribes
MIRNA genes into primary miRNA transcripts (pri-miRNAs),
which fold into stem-loop hairpin RNAs (Fig. 1a, left). In terrestrial
plants these primary miRNAs and subsequent pre-miRNAs are
processed by Dicer-like 1 (DCL1) to produce ~21 nt miRNAs.
Different miRNAs can then guide Argonaute 1 (AGO1) to either
target mRNAs for translational inhibition or guide cleavage of
mRNAs [16–18]. A separate pathway produces tasiRNAs from
Pol II-transcribed TAS genes (Fig. 1a, right). TAS transcripts are
targeted by miRNA-guided binding/cleavage events (not shown)
that trigger the production of dsRNA by RNA-dependent RNA
polymerase 6 (RDR6). Dicer-like 4 (DCL4) processes these
dsRNAs into 21 nt siRNAs, which can target AGO1 to complementary mRNAs and thereby regulate development [7, 19, 20].
Transposable elements (TEs) constitute a large percentage of
many plant genomes, sometimes exceeding 80% in a given crop
species, such as maize [21]. In terrestrial plants, a class of ~24 nt
siRNAs silences TEs via RNA-directed DNA methylation
[22]. This process initiates with the recruitment of RNA polymerase IV (Pol IV) to a locus (Fig. 1b). Pol IV is an enzyme related to
Pol II but with distinct protein subunits, domains, and motifs
specialized for its function in TE silencing [6, 23–26]. Pol IV
transcribes chromosomal TEs and DNA repeats, channeling short
non-coding RNAs to RNA-dependent RNA polymerase 2 (RDR2)
[27, 28]. RDR2 then produces a second RNA strand, releasing
~30 bp dsRNAs that are processed by Dicer-like 3 (DCL3) [28–
30]. Resulting 24 nt siRNAs guide AGO4-clade proteins to sites of
RNA polymerase V transcription, leading to DNA methylation by
Domains Rearranged Methyltransferase 2 [31]. Pol IV-dependent
DNA methylation is accompanied by repressive histone modifications that silence TEs and protect genome integrity [23, 32–34].
TEs can be activated in response to environmental stresses
[35, 36]. For example, the Arabidopsis thaliana LTR retrotransposon ONSEN is activated under acute heat stress conditions [33, 37,
38]. After many plant generations exposed to selective pressure,
retrotransposition events may sometimes enable an adaptive stress
response. However, they more frequently cause deleterious loss-offunction alleles or ectopic expression of genes. Detecting and
annotating TEs is therefore an important, recurring task in functional and comparative genomics [21, 39, 40]. Because the majority of siRNA clusters overlap with TEs in plant genomes, small RNA
sequencing (small RNA-seq) can assist in the TE annotation
388
Marcel Bo ¨ hrer et al.