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doi:10.1093/bfgp/els050
Processing of plant microRNA
precursors
Nicola¤s G. Bologna, Arnaldo L. Schapire and Javier F. Palatnik
Advance Access publication date 11 November 2012
Abstract
MicroRNAs are endogenous small RNAs known to be key regulators of gene expression in animals and plants.
They are defined by their specific biogenesis which involves the precise excision from an imperfect fold-back
precursor. These precursors contain structural determinants required for their correct processing. Still, there are
significant differences in the biogenesis and activities of plant and animal microRNAs. This review summarizes
diverse aspects of precursor processing in plants, contrasting them to their animal counterparts.
Keywords: microRNA; precursor; structural determinants; biogenesis; processing; Arabidopsis thaliana; plants; animals
INTRODUCTION
Small RNAs (sRNAs) are major regulators of gene
expression in animals and plants. They are usually
19–24 nt long and processed from double-stranded
RNAs (dsRNAs) by Rnase-III enzymes [1, 2]. Plant
sRNAs can be classified into different groups such as
microRNAs (miRNAs), small interfering RNAs
(siRNAs), trans-acting siRNAs, natural antisensesiRNAs and the recently discovered diRNAs,
which are generated after DNA lesions [3–6].
Although each class possesses its own characteristics
in terms of their biogenesis and size, all of them are
ultimately incorporated into RNA silencing complexes, which contain a member of the
ARGONAUTE (AGO) family as a main component [3]. The sRNAs guide AGO proteins to
target RNAs using base complementarity as a
search tool. In turn, AGO complexes regulate gene
expression at transcriptional or post-transcriptional
level [2].
MiRNAs are an appealing group of sRNAs, as
they originate from endogenous loci and regulate
other target RNAs than those generating the
sRNA [2, 7]. Plant miRNAs are usually around
21–22 nt long and mediate gene silencing at
post-transcriptional level [8, 9], which entails the (i)
endonucleolytic cleavage (slicing) and/or (ii) translational repression of a target mRNA. The importance
of miRNA-mediated regulation is evident by the
severe pleiotropic phenotypes in miRNA-deEcient
mutants [10–13].
The miRNA-binding sites in target RNAs usually
have an extensive sequence complementarity to the
miRNAs and most experimentally validated targets
contain five mismatches or less [14–16]. MiRNAs
that are conserved during evolution generally regulate transcription factors that control plant development [17]. Plant miRNAs regulate few targets,
usually duplicated members of one protein-coding
gene family, yet, disruption of this regulation
causes severe defects [2]. In contrast, animal
miRNAs regulate many target genes through the
base pairing of their 6-nt seed region [18].
However, the biological role of this regulation is
unclear in most cases.
Although miRNAs mediate diverse aspects of development and physiology in both plants and animals, there are substantial differences between them,
Corresponding author. Javier F. Palatnik, IBR (Instituto de Biologı́a Molecular y Celular de Rosario), CONICET and Facultad de
Ciencias Bioquı́micas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina. E-mail: [email protected]
Nicola¤s G. Bologna, PhD, was a graduate student at the IBR, and is currently a postdoctoral fellow at the ETH, Switzerland. His
research focuses on studying microRNAs in plants.
Arnaldo L. Schapire, PhD, is a postdoctoral fellow at the IBR Institute in Rosario, Argentina. His research focuses on studying the
mechanisms of microRNA processing in plants.
Javier F. Palatnik, PhD, is a group leader at the IBR Institute in Rosario, Argentina. His research group studies different aspects of the
biology of microRNAs.
ß The Author 2012. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]
38
Bologna et al.
suggesting they have appeared independently in the
two lineages [19]. Still, plant and animal miRNAs
are processed from precursors containing imperfect
stem–loops and generally regulate longer target
mRNAs in both systems. Here, we focus on the
processing of plant miRNA precursors and compare
them with their animal counterparts.
MiRNA BIOGENESIS PATHWAY: AN
OVERVIEW
Most plant miRNAs are generated from their own
transcriptional units. They are transcribed by RNA
polymerase II and then capped, spliced and polyadenylated [20, 21]. Although many animal miRNAs
are derived from introns or untranslated regions of
coding messages or primary transcripts containing
tandem precursors [22, 23], most plant miRNAencoding loci comprise independent, non-proteincoding transcription units [24]. However, there are
some known examples of transcripts harboring
tandem precursors in plants [25, 26] and precursors
located in mRNA untranslated regions [27].
The miRNA primary transcripts contain an internal stem–loop secondary structure (miRNA precursor) with the miRNA located in one of the arms.
The miRNA processing machinery recognizes structural determinants in the miRNA precursors and
produces staggered cleavages in the dsRNA, separated 21 nt of each other. These cuts release the
miRNA together with the opposing fragment of the
precursor that is interacting with it, called miRNA*
(Figure 1A).
The core component of the miRNA processing
machinery in Arabidopsis is DICER-LIKE1 (DCL1),
which is the RNAse type III that produces all cuts in
the miRNA precursors [10, 11, 28]. DCL1 is assisted
by the dsRNA-binding protein HYPONASTIC
LEAVES1 (HYL1) [29, 30] and the C2H2
zinc-finger protein SERRATE (SE) [31, 32]
(Figure 1). Both HYL1 and SE, improve the efficiency and precision of cleavage by DCL1 [33].
Although not lethal, hyl1 null mutations severely
impair miRNA maturation, which in turn causes developmental defects [30, 34, 35]. Mutations in SE
accumulate high levels of miRNA primary transcripts and less mature miRNAs [31, 32]. These mutants exhibit also general mRNA splicing defects
[36]. ABH1/CBP80 and CBP20, which encode subunits of the nuclear CBC also display general mRNA
splicing defects and accumulate more miRNA
primary transcripts [36, 37]. Thus, dual roles in splicing and miRNA processing distinguish SE and
CBC from HYL1, which is more specialized in
miRNA biogenesis.
DCL1 and HYL1 co-localize in subnuclear regions that have been termed dicing bodies or
D-bodies [38–40]. Analysis of miRNA primary transcripts revealed that they are also recruited to these
bodies, suggesting that these regions function as
miRNA-processing centers in plants [38, 39]. The
DCL1-/HYL1-containing regions have similar features as the Cajal bodies, which are known to participate in RNA metabolism (reviewed in [41]).
However, while the DCL1-/HYL1-containing
bodies contain SmD3 and SmB [39], they do not
express coilin [38, 39, 42], a main marker of the
Cajal bodies [43].
DAWDLE, a DCL1 interacting protein, is
thought to stabilize miRNA primary transcripts
until they are processed by DCL1 [44]. HASTY, a
homolog of the animal Exportin5, also contributes to
the levels of certain miRNAs [45]. However, whereas Exportin5 transports animal pre-miRNAs to the
cytoplasm (see below), the molecular role of
HASTY is unclear as all processing steps occur in
the plant nucleus. Still, HASTY might be associated
with other cargo, such as the miRNA/miRNA*.
Recently, it has been reported that the lack of
TOUGH, an RNA-binding protein, reduces the accumulation of miRNAs and siRNAs in vivo [46].
TOUGH binds to miRNA precursors in vivo and
might aid in the recruitment of primary transcripts
or contribute to the efficiency of the DCL1 processing complex. Finally, after the processing of the precursors, the miRNA/miRNA* duplex is released.
The 30 -ends of both miRNA and miRNA* are
20 -O-methylated by the nuclear protein HEN1, preventing its degradation [47, 48] (Figure 1A).
In contrast to plants, miRNA biogenesis is compartmentalized in animals. First, primary transcripts
are trimmed in the nucleus to separate the stem–
loop precursor from the rest of the transcript. This
process is achieved by a Microprocessor complex
formed by an RNase III-like enzyme termed Drosha
and the dsRNA-binding protein DiGeorge syndrome
critical region gene 8 (DGCR8; Pasha in Drosophila
melanogaster and Caenorhabditis elegans) [49–51]. The
fold-back precursors are then translocated into the
cytoplasm by Exportin 5 [52, 53]. Once there, these
stem–loops are cleaved by a Dicer, releasing 21-nt
miRNA duplexes [54–56] (Figure 1B). Dicer acts in
Processing of plant microRNA precursors
39
Figure 1: Overview of miRNA processing pathways in plants and animals. (A) Plant miRNA primary transcripts
are stabilized by DAWDLE (DDL). In the D-bodies, DCL1, assisted by HYL1, SE and nuclear CBC cleaves the precursor to release the miRNA/miRNA* duplex. The sRNAs are stabilized by the addition of a methyl group (black dot)
by HEN1. One strand of the duplex, the miRNA (red), is incorporated into an AGO protein complex. (B) In animals
(humans), the initiation step (cropping) is mediated by the Drosha ^DiGeorge syndrome critical region gene 8
(DGCR8) complex (also known as the Microprocessor complex) that generates pre-miRNAs. Then, the
pre-miRNA is recognized by the nuclear export factor Exportin 5 (EXP5) and is exported to the cytoplasm. The
cytoplasmic RNase III Dicer catalyzes the second processing step to produce miRNA duplexes assisted by TRBP or
PACT. One strand of the duplex is finally incorporated into an AGO protein.
concert with other dsRNA-binding proteins:
Loquacious, that contains three dsRNA-binding domains (dsRBDs) [57–59] in flies, and HIV trans-activator RNA-binding protein (TRBP) [60, 61] and
protein activator of the interferon induced protein
kinase (PACT) [62] in humans. In contrast to plants,
animal miRNAs are usually not methylated. Still,
methylation by HEN1 orthologs has been observed
in other classes of sRNAs, such as the single-stranded
piRNAs and certain siRNAs [48, 63–65].
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Bologna et al.
Structural determinants of miRNA
precursors
The processing of the fold-back precursors by the
RNAse III complexes causes the release of
miRNA/miRNA* duplexes (Figure 1). Although
the miRNA is incorporated into an AGO complex,
the miRNA* is generally degraded. The precision
in the position of the cuts along the precursor is of
key importance, as they determine the sequence of
the miRNA and therefore its target specificity. Of
special importance is the selection of the position for
the first cleavage reaction because the second cut is
usually performed by measuring a fixed distance
from the end of the precursor. The evidence accumulated so far, indicates that the secondary structure
of the fold-back precursor directs the activity of the
processing machinery represented by Drosha and
DCL1 complexes. In some, the biogenesis of the
miRNAs could proceed by other pathways. For example, a few recently evolved miRNAs have been
shown to depend on DCL4 rather than DCL1 in
Arabidopsis thaliana [27] and the activity of Drosha
can be bypassed by the splicing machinery, generating mirtrons [66, 67], which have been also
described in plants [68, 69]. The biogenesis of most
animal and plant miRNAs, however, is canalized
through Drosha and DCL1, respectively.
Animal precursors
A typical miRNA precursor in animals comprises a
stem of approximately three helical turns (65 nt), a
terminal loop and long single-stranded RNA
(ssRNA) Fanking sequence below the fold-back
structure (Figure 2A). The stem corresponds to the
miRNA/miRNA* plus an additional 11 nt of a
lower stem located below the miRNA.
Experiments performed in vitro have shown that the
terminal loop is dispensable for processing
the miRNA precursors, whereas the region below
the miRNA is essential [70, 71]. Furthermore, it
was demonstrated that the distance from the
ssRNA basal segments to miRNA/miRNA*
duplex is critical for cleavage site selection [70].
The model for the biogenesis of animal miRNAs
suggests that DGCR8/Pasha recognizes the precursor by anchoring at the ssRNA–dsRNA junction
while interacting with the precursor stem. After
this initial recognition, the processing center of
Drosha might be located, performing the first cleavage 11 bp away from the ssRNA–dsRNA junction
and releasing the fold-back from the rest of the
Figure 2: Structural determinants in animal and plant
miRNA precursors. (A) A typical miRNA precursor in
animals comprises a stem of approximately three helical
turns (miRNA/miRNA* duplex plus a lower stem), a
terminal loop and long ssRNA Fanking sequences.
(B) A plant miRNA precursor could be divided into
four parts comprising a lower stem, the miRNA/
miRNA*, an upper stem and the terminal loop. Many
plant miRNA precursors have a lower stem of 15 nt
below the miRNA/miRNA* that is followed by a large
bulge. (C) The current canonical ‘base-to-loop’ processing model in plants implicates an initial cleavage event
at the base of precursor followed by a second cut produced at 21-nt distance of this first cut close to the terminal loop. The miRNA/miRNA* is indicated in bold
lines.
transcript [23, 70]. The second cleavage can then
be performed by Dicer in the cytoplasm by recognizing the free 30 -end of the precursor stem and cutting 22 nt (approximately two helical turns) from this
end [57].
Plant precursors
In contrast to the defined in vitro systems used to analyze miRNA biogenesis in animals (e.g. [70, 71]),
most of the studies carried out in plants used transgenic plants expressing miRNA precursors [72–74].
In many plant precursors, a single change in the
lower stem of 15 nt below the miRNA is sufficient
Processing of plant microRNA precursors
to completely abolish its processing [72–75].
A random mutagenesis approach performed on
miR172a precursor revealed that point mutations
located in the lower stem significantly affected its
processing, whereas mutations located in the terminal loop were largely neutral [72]. Interestingly,
these unbiased mutations that affected miRNA processing identified by this approach also destabilized
the precursor structure [72].
A systematic analysis of plant precursors revealed
that many of them have a lower stem of 15 nt
below the miRNA/miRNA* that follows the presence of a large bulge [72–74] (Figure 2B). The current model implicates the recognition of this lower
stem to position the initial DCL1 cleavage event.
The second cut is produced in the stem at 21 nt of
this first cut. These structural determinants are similar
but not identical to those identified in animal
miRNA precursors (Figure 2C).
Loop-to-base processing of plant precursors
The animal stem–loop structures of miRNA precursors have highly uniform sizes of 65 nt, whereas in
plants there is a wide range of precursors from 50 to
>500 nt [76] (Figure 3, inset). This heterogeneity in
the precursor structures might also reflect differences
in the processing pathways. The precursors of
miR159 and miR319 are among those with long
fold-backs [77–79].
Detailed mutagenesis studies revealed that the
processing of these precursors begin with a cleavage
next to the loop [80]. DCL1 then continues to cut
the precursor three more times at 20- to 22-nt intervals in a ‘loop-to-base’ direction, until the miRNA is
finally released [80, 81] (Figure 3). Removing the
lower stem below the miRNA/miRNA* did not
affect miR319 processing. On the contrary, modifying the upper part of the precursor severely impaired
the biogenesis of miR319 [80].
The processing of plant precursors by multiple
DCL1 cuts has been also observed in other cases
[28, 82] and so far, is a distinctive feature not present
in animal precursors. In principle, the processing of
miR319 and miR159 precursors might lead to the
accumulation of several miRNAs. The experimental
evidence so far indicates that miR319 and miR159
have clear biological roles in plant development [78,
80, 83–85]. In turn, miR319 and miR159 are highly
conserved across the plant kingdom and copies of
miR319 can even be found in mosses, also being
41
processed in a loop-to-base direction, indicating
their ancient origin [80, 81, 86, 87].
The other sRNAs can be detected in vivo associated with AGO proteins [88]; however, their biological function remains to be elucidated. The
secondary structure of these long precursors contains
information regarding the final level of the different
potential sRNAs [80]. It seems that the presence of
large number of bulges in certain regions of the stem
prevents the accumulation of those sRNAs [80].
Young miRNAs
It was found that some newly evolved miRNA
fold-backs in plants show complementarity with
their target genes that extended beyond the
miRNA/miRNA* region [24, 27, 89–91]. This observation strongly suggests that many young MIRNA
genes derive from inverted duplication events
of their targets, giving rise to ‘proto-miRNAs’
[24, 27, 89–91].
However, these newly formed RNA structures
do not necessarily have the requirements for
miRNA biogenesis as described above and some of
them might be processed by different DCLs (reviewed in [2]). In this model, the dsRNA with perfect base pairing formed after a gene duplication
event is initially processed by DCL3 and DCL4 to
release several siRNAs (or proto-miRNAs). During
evolution, the accumulation of mutations in the
fold-back arms would lead to changes in the secondary structure and shortening of the hairpin stem, and
finally, the processing will be channeled through the
miRNA-specific DCL1.
In accordance with this hypothesis, mir822,
miR839 and miR869 fold-backs are processed by
DCL4 rather than DCL1 [92, 93]. DCL3 has also
shown to process conserved miRNAs of Arabidopsis
[94], as well as certain precursors in rice to yield
long-miRNAs (24 nt) [95]. For example, in
Arabidopsis the activity of DCL3 causes the accumulation of a 24-nt species for certain miRNAs in addition to the main 21-nt class. This is most obvious in
inflorescences, which is also the tissue where DCL3
transcripts accumulate [94]. The processing of
miR1850 in rice leads to 21- and 24-nt miRNA
species by the sequential action of DCL1 and
DCL3, respectively. Furthermore, DCL1 and
DCL3 can act in parallel on several other precursors
as well [95]. It is certain that these recently evolved
miRNAs contribute to the heterogeneity of plant
precursors (Figure 3, inset).
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Bologna et al.
Figure 3: The proposed model for the biogenesis of miR319 and miR159. The miRNA precursor is first cleaved
below the loop by DCL1. The processing then continues with three more cuts until the miRNA/miRNA* is released
(indicated in bold lines). The miRNA is subsequently incorporated into an AGO complex. Inset: size distribution of
the fold-backs of C. elegans and A. thaliana. The miRNAs from Arabidopsis are further classified into young and evolutionary conserved miRNAs (at least in angiosperms). The precursors (pre-miRNAs) were considered as the sequence between the miRNA and the miRNA*.
PERSPECTIVES
Plant miRNA precursors are much more heterogeneous than their animal counterparts. In part, this
heterogeneity resides in the base-to-loop and
loop-to-base processing mechanisms that generate
them, as well in the multiple cuts that can be produced by DCL1 to release the miRNAs. Young
miRNAs, formed by inverted duplication of
protein-coding sequences, contribute to the large
variation of shapes and sizes of the plant precursors.
Still, a more systematic approach is needed to understand the extension of the complexity of miRNA
processing in plants. Interestingly, it has been recently shown that precursors containing a structural
asymmetry due to the presence of bulges in either
the miRNA or the miRNA*, will then generate
sRNAs capable of transitivity [96]. It has been suggested that AGO1 complexes with these sRNAs
recruit RDR6 and SGS3, which in turn generate
dsRNA from their targets and secondary siRNAs
[96], therefore, linking the processing of the
miRNAs with their activity. It would be interesting
to determine the molecular mechanisms and the
causal relationships between the processing of the
precursor and the activity of the sRNAs.
Key Points
Processing of plant microRNA precursors.
Comparison of animal and plant microRNA biogenesis.
Structural determinants of animal and plant microRNA
precursors.
FUNDING
Grants from the Agencia Nacional de Promoción
Cientı́fica y Tecnológica (ANPCyT) and Consejo
Nacional de Investigaciones Cientı́ficas y Técnicas
(CONICET) (to J.P.).
Processing of plant microRNA precursors
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