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REVIEW ARTICLE

Journal of

Biogenesis, Functions and Fate of


Plant microRNAs

Cellular
Physiology

AFSAR RAZA NAQVI,1,2 MARYAM SARWAT,1,3** SHIRIN HASAN,4,5


1
AND NIRUPAM ROYCHODHURY *
1

Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg,

New Delhi, India


2

Department of Periodontics, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina

Pharmaceutical Biotechnology, Amity Institute of Pharmacy, Amity University, Noida, India

Faculty of Life Sciences, Department of Biochemistry, A. M. University, Aligarh, Uttar Pradesh, India

Department of Biochemistry, Duke University, Durham, North Carolina

microRNAs (miRNAs), a recently discovered class of small RNAs, are endogenously transcribed non-coding RNAs that are known to
control diverse developmental processes and defense responses. They regulate these pathways by ne-tuning the levels of transcripts to
which they bind and cause their cleavage or translation repression. Several studies on the processing of miRNA precursors have shed light
on the essential structural features for precise release of miRNA duplexes. The identication of a protein that degrade single stranded
small RNA has provided us with some understanding of how miRNA ux is maintained in plants. This review focuses on the genome
organization, biogenesis, miRNA activity, and the fate of miRNAs.
J. Cell. Physiol. 227: 31633168, 2012. 2012 Wiley Periodicals, Inc.

microRNAs (miRNAs) are endogenously transcribed single


stranded non-coding RNA species that are 21 nt long. These
are present in all eukaryotes and similar small RNA molecules
are also being reported from prokaryotes. miRNAs exhibit
evolutionary conservation and like any protein coding gene,
contribute toward the diversication of species along the
evolution. The rst miRNA to be discovered was lin-4 in
C. elegans by Lee et al. (1993) and were termed short temporal
RNAs. They observed a 22 nt small RNA sequence generated
from a precursor of lin-4 gene can control the expression levels
of LIN-14 protein. The lin-14 transcript was later found to
possess several complementary binding sites for lin-4 sequence
on its 30 -UTR. After almost 7 years, another small temporal
RNA, namely let-7 was identied in nematode that was
conserved across animals and was found to regulate protein
levels of various transcripts due to its binding potential at the
30 -UTR of these targets (Pasquinelli et al., 2000; Reinhart et al.,
2000). On the other hand, in plants, the initial cloning led to the
identication of various miRNAs families (miR156, miR159,
miR164, miR171, etc.) that were later found to be highly
conserved across the plant kingdom (Reinhart et al., 2002).
Hundreds of miRNAs are reported in animals and plants,
however, there is no sequences conservation except for
miR854 and miR855 (Arteaga-Vazquez et al., 2006). Both,
miR854 and miR855 were found to bind and regulate levels
of OLIGOURIDYLATE binding PROTEIN1b (At UBP1b)
transcript. In animals, miR854 is generated from intergenic
region and regulate the translation of a transcript that performs
similar functions as in plants. This is a unique example of
conserved miRNA with same sequence and functionality with
conservation across plant and animals.
Plants and animals share several similarities in the basic
phenomenon underlying miRNA biogenesis and its
functionality, nonetheless, there are still unique features
attributed to the generation of these regulatory RNA
molecules. In this review we have focused on the recent
advancements in our understanding of miRNA processing
and fate of miRNAs while comparing the features of this
phenomenon in animals.
2 0 1 2 W I L E Y P E R I O D I C A L S , I N C .

Plant miRNAs
Origin of miRNA

The miRNA genes are thought to be evolved from the


duplication of certain genomic regions that encodes proteincoding RNA (Allen et al., 2004; Fahlgren et al., 2007). These so
called old miRNAs are expressed in high quantities and were
observed to be present on multiple loci on genome. However,
the recent deep sequencing analysis suggests that newly
identied miRNAs, similar to animals, are likely to have
come into existence by random acquisition of complementary
sequences and that they are not functionally relevant, as
observed in neutral evolution (Fahlgren et al., 2010). Such
observations that miRNA-generating precursors may arise
from diverse genomic location refutes the previous universal
duplication hypothesis. These young miRNA genes are
believed to come under the transcription activity of promoter
and thus becoming active (de Felippes et al., 2008). The low
amounts of the young MIR gene may be responsible for the
reduced off-target effects of these sequences and, in turn, avoid
generation of siRNAs. Alternatively, the transposable regions
within genome may also act as reservoir to generate fold-back

Department of Periodontics, School of Dentistry, University of


North Carolina, Chapel Hill, NC. E-mail: afsarrazaa@yahoo.co.in
*Correspondence to: Nirupam Roy Choudhury, Plant Molecular
Biology Group, International Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
E-mail: nirupam@icgeb.res.in
**Correspondence to: Maryam Sarwat, Pharmaceutical
Biotechnology, Amity Institute of Pharmacy, Amity University,
Noida-201303, India. E-mail: maryam21_7@yahoo.com
Manuscript Received: 14 September 2011
Manuscript Accepted: 6 January 2012
Accepted manuscript online in Wiley Online Library
(wileyonlinelibrary.com): 17 January 2012
DOI: 10.1002/jcp.24052

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structures which eventually assume pre-miRNA-like secondary


structures (Piriyapongsa and Jordan, 2008).
Expression and Genomic organization of miRNA genes

MiRNAs are encoded from a locus under the inuence of their


own promoter. Similar to other protein-coding genes, the
miRNA generating genes are controlled by the activity of
various transcription factors. It is now well demonstrated that
miRNA genes respond to external stimuli including abiotic
(drought, temperature, salinity, etc.,) and biotic (bacteria,
viruses, etc.,) components and that leads to the modulation
of the expression levels of the mature miRNAs (Reviewed by
Naqvi et al., 2010; Naqvi et al., 2011a; Our unpublished data).
Evidently, this feature suggests that while miRNAs regulate
several genes they themselves are controlled by transcription
factors. MiRNA-encoding genes exhibit both temporal and
spatial expression patterns. It is also hypothesized that miRNA
promoters may have acquired gene-specic cis-regulatory
elements which may govern unique expression of miRNA.
Some miRNA are ubiquitous yet others may dene cell lineage
and thus their expression is restricted to a particular cell
type. For instance, a recent high throughput sequencing of
Arabidopsis root tissues has revealed that miR169 and miR162
families are present across all cell types, miR156 and miR157
exhibited high variance (Breakeld et al., 2012).
Unlike in animals, the miRNA genes in plants are generally
monocistronic. However, there are few plant miRNA families
that are transcribed as polycistronic units. These include
miR156, miR166, miR167, miR395, etc., gene clusters where
two or more members reside on same transcript (Chuck et al.,
2007; Wang et al., 2007; Zhang et al., 2009). Amongst these,
while miR166 cluster is conserved across plants, miR156 and
miR395 are well characterized only in case of monocots. In
maize, miR395 gene clusters are present on four different loci
each encoding three to ve genes (Zhang et al., 2009). MiRNA
genes are usually located either within intergenic regions or in
unannotated loci (Rajagopal et al., 2006). However, there are
still some miRNAs originating from exons of protein-coding
genes (Zhang et al., 2009).
Plant miRNAs, similar to animals, are transcribed by RNA
polymerase II to generate primary miRNA (pri-miRNA)
transcripts. The pri-miRNAs (pri-miRs) have characteristic
features of 50 cap, 30 -poly A and intron splicing (Bartel, 2004;
Xie et al., 2005; Fig. 1). The lengths of these pri-miRs are variable
and range from few hundred bases to several kilo bases. For
instance, the RACE analysis of a huge set of pri-miRs (in Maize)
has shown that the size of pri-miRs vary from 250 to 2,000 bp
(Zhang et al., 2009). Similar to the protein coding genes, these
transcripts possess TATA-box sequences (at 23 to 28
positions). It was also noticed that the introns in pri-miRs
are prevalent in the 30 region and have canonical GU-AG sites
for splicing.
Several animal miRNAs originate from intronic loci and
these are processed through mirtron pathway which is
independent of DROSHA/PASHA activity (Ruby et al., 2007;
reviewed in Naqvi et al., 2009). In plants, DCL1 orthologs
generate one such intronic miRNA and which might perform
feedback mechanism (Rajagopal et al., 2006; Axtell et al., 2007).
However, the mechanistic aspect of the intronic miRNAs
generation is yet to be elucidated in plants.

Fig. 1. microRNA biogenesis in plants. miRNAs are transcribed


from endogenous genes as pri-miR by RNA polII. These transcripts
have characteristic features of RNA polII activity including
5(-methylated cap and 3(-poly (A) tail. The local hairpin structure
within pri-miR is recognized by DCL1/HYL1/Serrate proteins to
generate precursor miRNA (pre-miR). DCL1 acts on pre-miR and
release miRNA duplexes. These duplexes are modied by HEN1
that methylates 3(-OH residues. HASTY, a homolog of Exportin-5,
exports out modied miRNA duplexes to cytoplasm. These duplexes
further recruit several proteins, including AGO1, to form a functional
miRNA-protein complex termed miRNA-induced silencing complex
(mi-RISC). The mi-RISC assembly binds to target transcripts and
leads to either cleavage or translational repression. The single
stranded miRNAs that lack any methylation or uridylation are
degraded by SDN1 protein.

microRNA biogenesis

miRNA biogenesis is a complex process and is


compartmentalized among nucleus and cytoplasm. So far,
several proteins have been identied as processing factors
that yield mature miRNAs and the list is expected to increase in
near future. The miRNA biogenesis is discussed below under
two subsections, namely, nuclear, and cytoplasmic processing.
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Nuclear processing

The local secondary structure contained within the pri-miRs


is recognized by RNA binding proteins namely DCL1/HYL1/
Serrate (Han et al., 2004; Lobbes et al., 2006). The
endonucleolyic activity of these proteins at precise location

PLANT microRNA BIOGENESIS

release stemloop region from the parent pri-miR (discussed in


detail in subsequent section; Fig. 1). This stemloop structure is
called precursor-miRNA (pre-miRNA). The remaining part of
the pri-miR will be acted upon by exonucleases and eventually
degraded. The siRNA processing in Arabidopsis occurs in special
structures called Cajal bodies which also colocalize with the
components of miRNA machinery viz., DCL1/HYL1/SE1
(Pontes et al., 2006; Pontes and Pikaard, 2008). However,
yet another complex, different from that of Cajal bodies and
comprise of Dicer-like 1 (DCL1) and Hyponastic Leaves1
(HYL1) proteins have also been identied in the perinuclear
region and is termed D/SmD3 bodies (Kurihara et al., 2006;
Fang and Spector, 2007; Fujioka et al., 2007; Song et al., 2007; Liu
et al., 2012). These two bodies may exhibit similar nuclear
location and size but D/SmD3 bodies lacks coilin protein which
is considered as a marker for Cajal bodies. It was demonstrated
that DCL1/HYL1 containing bodies associate with pri-miRNAs
and thus participate in pri-miRNA processing. However,
authors also proposed that these structures may act as
assembly and storage sites for miRNA processing components
(Song et al., 2007).
Similar to the pri-miRs, the pre-miRNAs (pre-miR) are also
highly variable in their sizes (Zhang et al., 2006). Studies by
Bologna et al. (2009) have shed light on the mechanism of
processing of plant stemloop structures. Further, the
structural and sequence requirement for the precise release of
miRNA duplexes from the pre-miRs were also demonstrated
(Mateos et al., 2010; Song et al., 2010; Werner et al., 2010).
Together, these results rened our understanding on miRNA
biogenesis and are discussed in the subsequent sections. In
brief, the cleavage of pre-miR to miR/miR duplex by DCL1 and
other accessory proteins may take place in two different
directions. While most of the plant pre-miRs (miR172, miR398,
etc.) follow stem-to-loop processing (Werner et al., 2010),
pre-miR319 and 159 are processed in reverse orientation,
i.e., loop-to-base processing (Bologna et al., 2009).
Sequence determinants of pre-miRNA processing

The observations that mutations in pre-miRs (either in miR/


miR region or in their vicinity) may lead to altered miRNA
biogenesis opened a new scope to study the role of structural
determinants of the pre-miRs. In animals, this mechanism was
investigated relatively earlier in lieu of the uniform size and
structure of pre-miRs (Han et al., 2004). They came up with a
11 rule which suggests that the rst cleavage is made below
the miR/miR duplexes after sensing the 11 nt (one helical turn)
from the proximal stemloop junction. However, due to
existing variability of sizes among plant pre-miRs, the existence
of such generalized mechanism in plant systems is unlikely.
Recently, three independent groups studied the ner details of
the processing of various plant miRNA families and proposed a
general model encompassing desired features for the release of
miR/miR from their respective precursors (Mateos et al., 2010;
Song et al., 2010; Werner et al., 2010).
Stem-to-loop processing

Pre-miR processing in animals is well dened and is extensively


studied. In animals, pre-miRs fold in a similar fashion with 33
base pair long stem region and a terminal small loop (Fig. 2a).
The rst cut is mediated by Drosha at 11 nt away from the
base of the stem. Subsequently Dicer acts on the released
Drosha product and generate miR/miR duplex of 22 nts.
Most of the plant pre-miRs are processed similar to animals.
Pre-miR172a, pre-miR163 and few others were observed to
undergo two successive cleavages by miRNA processing
enzymes. A recent deep sequencing study by Breakeld et al.
(2012) shows that of the 149 of the 213 miRNA precursors
analyzed have features consistent with the stem-to-loop
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processing. Further supporting that major miRNA genes are


processed in this fashion. DCL1/HYL1 catalyzes the initial
cleavage which is made at the base of the stem (in animals this
step is mediated by Drosha). The released structure is cleaved
by DCL1 alone to generate miR/miR duplexes [Fig. 2b (i)]. The
physical mapping of the pre-miR172 processing was examined
by several groups and they concluded that initial cut by DCL1/
HYL1 is located 15 nt from the base of the stem. Pre-miR172
was used to study pre-miR biogenesis as the read out (early
owering) of Arabidopsis plants can be easily monitored. It was
observed that closing bulges immediately below the loop-distal
cleavage sites increased the accumulation of accurately cleaved
precursor miRNAs but decreased the abundance of the mature
miRNAs. A pri-miR variant with an unpaired lower stem was
not processed, and variants with a perfectly paired middle or
upper stem were processed normally. Werner et al. (2010)
demonstrated that mutations in the 50 -arm of the pre-miR172
(keeping the miR172 activity still intact) did not lead to any
signicant phenotypic alteration. It may be noted that the
mature miRNA resides in the 30 -arm of the pre-miR172.
Importantly, unlike animals, plants miRNA can tolerate
changes in the duplex region. This nding, however, could
be attributed to the fact that animal miR/miR exhibits lesser
complementarity than plants suggesting that plant miRNAs are
comparatively more elastic with respect to their processing.
Loop-to-base processing

Unlike other plant pre-miRs, miR159 and miR319 are


processed in altogether different orientation, i.e., the rst
cleavage initiates toward the loop region of pre-miR (Bologna
et al., 2009). Intriguingly, both of these pre-miRs have unusually
long double stranded backbone and are highly conserved across
plant kingdom. MiR319 over-expression leads to reduction
in Teosinte, Cycloidea, and PCF1 (TCP) transcription factor
family proteins which eventually leads to crinkled leaf
phenotype and was used as a read out to study the effect of
sequences/structures in the generation of mature miRNAs.
Figure 2b (ii) shows the secondary structure of pre-miR319.
The determinants for precise release of miR/miR duplex was
studied by deletion or introduction of point mutations across
the pre-miRs. While the deletion of lower stem region did not
have any effect on plant phenotype, the upper stem deletion led
to complete abolishment of the abnormal phenotype. This
clearly indicated that the upper stem is indispensable for the
generation of functional miRNAs. To elucidate the direction of
processing, a modied RACE strategy was employed, and it was
observed that miR319a precursor gave rise to four different
products, indicating that the processing must have initiated
from the terminal loop of the pre-miR319a. The most abundant
small RNA is evidently mature miR319, however, there are
other small RNA sequences already present in the Arabidopsis
small RNA libraries, albeit at relatively low frequency (Rajagopal
et al., 2006). The similar observation was made with the
miR319b and miR159a in Arabidopsis as well as in primitive plant,
moss.
Further, the outcome of deletion of 8 nt or larger region
from or closing the bulge of the pre-miR319 upper stem
indicated that any change in this region leads to adverse effect
on the formation of mature miRNA, suggesting the vital role
of upper stem in guiding precise cutting by DCL1. This was
evident from the observation that the plant expressing these
engineered constructs had cleavage sites in the middle of the
miR/miR duplex region. Interestingly, the change in the loop to
base and vice versa reverses the polarity of the miRNA, i.e., the
previously 50 strand would become 30 strand. However, this
does not compromise with the miRNA generation.
The pre-miR319a contains ve bulges beyond the miR/miR
region and the role of these bulges present across the

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Fig. 2. Schematic representation of pre-miR structure and unique features determining the pre-miR processing in (a) animals and (b) plants.
a: In animals, pre-miRs have highly uniform size and assume similar secondary structures. In a sequential manner, these are processed by two
endonucleases Drosha and Dicer. The rst cut is mediated by Drosha which is 11 nt from the base of stem. Dicer then cleaves the Drosha
product to release 22 nt miR/miRM duplex. b: On the basis of processing plant pre-miRs are classied into two distinct categoriesstemloop and
loopbase. (i) Those with short stemloop are generally processed through stem to loop and most of the plant pre-miRs studied are known to
be processed in this manner. This is similar to animal processing; however, the cleavage occurs at 15 nt from the stem terminal. (ii) Two plant
pre-miRs (pre-miR159 and 319) have long stemloop structures and the initial cut is located near terminal loop. The generation of miR/miRM
duplex require multiple (4) cuts by DCL1 before the release of miR/miRM duplex. The presence of bulges within and below the miR/miRM duplex
favors the generation of bonade mature miRNAs.

pre-miR319 were studied by closing individual bulges. It was


observed that only the bulge next to miR/miR (and to some
extent the next one) have pronounced effect on the production
of miR319 but not those beyond it. Also, when the bulge
immediate next to miR/miR was closed, the generation of an
altogether new small RNA species was noticed (Bologna et al.,
2009). This clearly indicates that besides acting as guide to
locate precise cuts generating mature miRNA, the upper stem
prevents accumulation of other small RNAs by incorporating
few bulges along the stemloop structure. Overall, these data
point toward a new mechanism of miRNA biogenesis, however,
very few pre-miRs follow such non-canonical processing.
The duplex miRNAs thus released from the pre-miRs, while
still in the nucleus, are modied at their 30 -hydroxyls by a
methylase, namely Hua Enhancer1 (HEN1; Yu et al., 2005). The
methyl moiety added to these duplexes confers subsequent
stability to the miRNAs, protects them from any other
enzymatic modication such as uridylation and prevent
miRNAs to prime the RNA dependent RNA polymerase
activity on certain target RNA to generate dsRNA (Li et al.,
2005; Yu et al., 2005; Fig. 1). This aspect is discussed in detail in
subsequent sections. Since miRNAs acts in a sequence specic
manner, any sequence modication may have profound
downstream effect(s). The methlyation of miRNA duplexes
may also act as an exit signal to the cytoplasm (Park et al., 2005).
A nuclear shuttle protein, HASTY1 (orthologue of Exportin 5),
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has been demonstrated to direct miRNA duplexes to the


cytoplasm. The Arabidopsis plant mutant for HASTY1 (HST1)
exhibits enhanced accumulation in the levels of pre-miRs. As in
case of other proteins involved in miRNA biogenesis, these hst1
plants exhibit developmental abnormalities. This clearly
suggests that HST1 is also an integral part of the miRNA
processing machinery. However, it was observed that the hst1
plants did not lead to complete abolishment of the mature
miRNA generation. This indicates that either there are certain
other proteins that may act redundantly on these miRNA
duplexes or given the fact that the miRNA duplexes are present
at high levels in the nucleus, the possibility of passive transport
across the nucleus cannot be ruled out.
Cytoplasmic processing

In the cytoplasm, the DCL1 associated duplex miRNAs recruit


another critical miRNA machinery protein called Argonaute-1
(Morel et al., 2002; Vaucheret et al., 2006). In plants, there are
several members of this Argonaute family, each involved in
some specic small RNA biogenesis pathway (Mi et al., 2008;
Montgomery et al., 2008). However, the roles of few members
of this family are yet to be deciphered. Nonetheless, there is
a plausibility of functional redundancy among these proteins.
For instance, the mutants of Ago1 and Ago10 display similar
developmental defects hinting at probable redundancy (Morel
et al., 2002).

PLANT microRNA BIOGENESIS

Of the 10 known Argonautes in Arabidopsis, Argonaute1 is


specically associated with miRNA biogenesis. It is proposed,
that the binding of AGO1 and other accessory factors
subsequently leads to the unwinding of miRNA duplexes
generating a guide strand (miRNA) and a passenger strand
(miRNA). The major governing factor in this process is the 50
sequence information of the miR duplex (Eamens et al., 2009).
As a general rule, the strand with lower thermodynamic
stability will be retained with AGO1 and subsequently, will
guide the functional protein machinery. This complex of
miRNA and AGO1 (and other accessory proteins) is termed
miRNA-induced silencing complex (mi-RISC) where AGO1
performs the slicer activity (Baumberger and Baulcombe,
2005). High-throughput sequencing of small RNA pool from
various plant systems has provided some insight into the aspect
of strand retention. Recently, it has been demonstrated that
50 -nucleotide of guide strand decides the sorting of miRNA to
RISC containing different AGOs (Mi et al., 2008; reviewed by
Naqvi et al., 2011a). By corollary, function of an miRNA may be
dened by the cellular expression and location of AGO to
which it is sorted.
Effects of miRNA binding to its target(s)

Once the functional mi-RISC is formed, the miRNA guides the


associated protein machinery to its cognate targets. In plants,
unlike in animals, one miRNA sequence binds to a limited
number of targets which can be attributed to the tight sequence
complementarity requirement exhibited by them. For a target
to be regulated by a miRNA, both of them should be expressed
in overlapping space and time. There are specic examples of
targets that can escape miRNA regulation by expressing either
at different time points or by expressing in mutually exclusive
tissues. Moreover, there are certain transcripts that can bind to
miRNAs but cannot be degraded thereby allowing certain other
targets to escape the miRNA-mediated regulation, a
phenomenon known as target mimicry (Franco-Zorrilla et al.,
2007). For instance, Induced by Phosphate Starvation1 (IPS1)
transcript has complementary sites for miR399 with a mismatch
in the central region (1113 nt) beyond the seed region (28 nts
from the 50 of mature miRNA). Although this allows miR399 to
bind IPS1, it is unable to cleave its target and thus acts as a sink
for the miRNAs. On the other hand, Pho2, the bonade target
of the miR399, escapes the suppression by miR399 even in case
the miRNA is present at high levels. When the sequences on
IPS1 were mutated such that the miRNA could bind to it with
perfect complementarity, it led to the degradation of IPS1
(Franco-Zorrilla et al., 2007). These results support the notion
that when several targets are present, some will be favored over
others and such phenomenon may be governed by the strength
with which miRNA binds to its target.
Fate of miRNA targets

In plants, generally, binding of miRNAs to their respective


targets leads to transcript cleavage (Palatnik et al., 2003). Global
proteomic analysis in Arabidopsis, however, has demonstrated
that while a major population of miRNAs is involved in target
degradation, a small set of most of them are also involved
in translational repression of similar targets (Brodersen et al.,
2008). A well established translationally repressed transcript
is Apetala-2 which is a target of miR172 (Chen, 2004).
In humans, additional dimension to miRNA-mediated posttranscriptional control has emerged from a recent nding
where miRNA binding was found to induce translation of target
genes under specic conditions (Vasudevan et al., 2007),
however, no miRNA has been reported to follow such pathway
in plants.
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microRNAs in defense pathways

Role of miRNAs in plant defense has been demonstrated in a


diverse class of pathogens. MiRNAs may serve as key signature
molecules in pathogen infections, especially those of viral origin.
For instance, Naqvi et al. (2010) demonstrated the levels of
miR319/miR159 and miR172 are induced in Tomato leaf curl New
Delhi virus (ToLCNDV) disease progression. The studies clearly
highlight the involvement of miRNAs in viral disease and the
potential to exploit these molecules for development of
antiviral strategies. The relevance of the miRNA deregulation
observed during infection is further substantiated by in silico
analyses where most of the ToLCNDV encoded ORFs were
found to possess putative host encoded miRNAs binding sites
(Naqvi et al., 2011b). Importantly, it was predicted that few
miRNA sequences could potentially target certain viral ORFs.
This observation is of signicant importance since no function
has been attributed to miRNA sequences in plants. However,
in animals, several lines of evidences support the role of
miRNA in regulating target transcript levels (Ghildiyal et al.,
2010). These ndings open up a new dimension to investigate
the host-virus arms race in plants.
Turn-over of miRNAs

MiRNAs, like other RNA molecules, are prone to degradation


by exonucleases and are required to bring about rapid changes
in transcriptome. Similar to proteins, where addition of a small
tag (ubiquitin) acts as a signal for degradation, the stability of
miRNAs is also governed by addition of certain groups. For
instance, addition of methyl group renders stability to miRNAs
while the uridylation of the mature miRNAs may act as a signal
for their degradation (Yu et al., 2005). However, the similar
addition of polyUs to miRNAs confers resistance to certain
exonucleases (Ramchandran and Chen, 2008). In plants,
uridylase has not been identied as yet. The characterization
of such proteins would provide meaningful details on existing
loop holes in miRNA homoestasis. Besides uridylation, the
polyadenylated miRNAs (intact or truncated) have also been
observed in deep sequencing of various plant tissues. The nontemplated addition of adenine-tail to miRNAs makes them less
susceptible to decay in presence of plant extract, indicating this
to be a likely mechanism contributing to the miRNA stability
(Lu et al., 2009). The animal miRNAs, on a similar line, are also
reported to be stabilized by addition of adenine residues at the
30 -hydroxyl groups (Katoh et al., 2009).
Recently, a protein identied by Ramchandran and Chen
(2008) has shed more light on our understanding of miRNA
ux. They have characterized a member of 30 to 50
exoribonuclease family protein, namely small RNA degrading
nuclease-1 (SDN1). The biochemical studies performed with
SDN1 indicates that this cannot act on DNA oligonucleotides
and failed to degrade miRNA in a miRNA/miRNA duplex.
Further, SDN1 degrades small RNAs within a size range of
1727 bp and yield a product of 89 nts long, suggesting that it
cannot act on molecules smaller than 8 nts. This degradation
was independent of the sequence of small RNA reecting that it
may act a predominant enzyme regulating miRNA steady state
levels. However, the presence of methyl group and poly U tail
deters the activity of SDN1 on miRNAs. Intriguingly, when
Arabidopsis plants were mutated for SDN1 gene, no signicant
changes were observed in the levels of miRNAs. This suggests
the redundancy among the members of this family. As expected,
the sdn1/sdn2 double mutants accumulated higher levels of
miRNAs compared to wild type plants. In Arabidopsis overexpressing an anti-miRNA (amiR) targeting exonuclease
domain of related members of SDN family, the miRNA levels
increased to even higher than that of individual or double SDN
mutants. These Arabidopsis plants display severe developmental
abnormality, as is observed in mutants of other miRNA

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processing enzymes. The recruitment of SDN1 to miRNAs


needs to be elucidated to bring a comprehensive picture of
how it regulates miRNA clearance. It is also important to
characterize other SDN family proteins in order to understand
their exact roles in small RNA or other related pathways.
Although the miRNA loading onto RISC is still awaits ner
details in plants, the similar processes have been extensively
studied in animal systems. Argonaute1 may also contribute to
miRNA turn-over simply by protecting miRNAs within protein
core and thereby making them non-available to exonucleases.
This is consistent with the observation that unlike other miRNA
processing factors, the ectopic expression of AGO1 may
have profound effects on miRNA accumulation (Vaucheret
et al., 2004). In animals, at least, AGO1 may preferentially
incorporate miRNAs that have readily accessible targets
instead of those with no targets available. Additionally, when
targets of a particular miRNA do not exist, AGO1 may release
miRNAs thereby making them susceptible to exonuclease
activity (Chatterjee and Grohans, 2009; Wang et al., 2009).
Acknowledgments

ARN is thankful to CSIR, India for Senior Research Fellowship.


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