1
Szarzynska B, Sobkowiak L, Jarmolowski A, Szweykowska-Kulinska Z
Department of Gene Expression, Faculty of Biology, Adam Mickiewicz University, Umultowska 89,
61-614 Poznan, Poland
Gene structures and processing of
plant pri-miRNAs
Res. Adv. in Nucleic Acids Research 1, 2011
SUMMARY
It has been shown that miRNAs play an
important role in posttranscriptional regulation
of plant gene expression. However, mechanisms
by which the expression of plant MIR genes is
controled remain to be discovered. In plants
miRNA-coding sequences are transcribed by
RNA Pol II and in terms of their processing (5'
and 3' end formation as well as splicing) MIR
transcripts show many similarities to mRNAs.
However our recent results support the notion
that miRNA primary precursors are recognized
by miRNA biogenesis machinery immediately
after transcription or even co-transcriptionally.
Therefore it seems very likely that the early steps
of miRNA biogenesis including transcription,
primary transcript processing and pri-miRNA
maturation show spatio-temporal overlap. In this
section we present an overview of data concerning
plant MIR gene organization and discuss
possible meaning of their features in very first
stages of miRNA biogenesis.
INTRODUCTION
Plant MIR genes are typically independent
transcriptional units (Chapter 1). The average MIR
gene length of several hundred bp is far less than
the average calculated for Arabidopsis and maize
protein-coding genes (1,2). Nevertheless, it seems
suprising that biogenesis of 21 nt-long miRNA
molecule is preceded by synthesis of the
precursor several hundred nucleotides in length.
Moreover, it has been shown that great part of
MIR genes contain introns and that their primary
transcripts undergo splicing, though it is not clear
whether this process is necessary for proper
miRNA maturation. It has been also found that
some of the pri-miRNAs may undergo alternative
splicing. However functional meaning of the
observed pri-miRNA diversity remains elusive. On
the other hand, there are some examples of
miRNA-coding sequences embedded within
introns of previously characterized genes (Chapter
2). Aditionally, MIR loci partially overlapping
known genes localized both in the same and in
2 Szarzynska B. et al
the opposite orientations have been identified.
Studies on the potential crosstalk between
processing of pri-miRNAs and transcripts
originating from the same loci may reveal another
level of gene expression regulatory network.
1. Plant intergenic MIR loci
1.1 MIR gene organization
Metazoan miRNA-coding sequences are
found mainly within introns and exons of
previously characterized genes (3,4). In contrast,
plant MIR genes are typically independent
transcriptional units (5). Based on up-to-date
results, lengths of plant MIR genes range from
around 250 bp to over 3000 bp (6,7). It has been
observed that regardless MIR gene length and
exon/intron structure, distance from transcription
start site to pre-miRNA-coding sequence is
usually shorter than 3' MIR region (measured from
pre-miRNA-coding sequence to the 3' boundary
of the MIR gene). The alignment of the established
A.thaliana and Z.mays pri-miRNA sequences to
genomic sequences revealed that some of the
plant MIR genes may contain introns (6-14). It
has been found that miRNA and miRNA* are
typically encoded by the first exon, however in
the case of A.thaliana MIR156a gene miRNA-
coding sequence is embedded in the second exon
and in the case of A.thaliana MIR172b it is
localized within the third exon (6). 5' and 3' splice
sites of all identified introns are in concordance
with the consensus sequences established for
plant U2-dependent intron type (‘GT...AG’ class)
(Figure 1). Lengths of the identified introns vary
form several dozens (e.g. 86 bp in the case of
MIR171b A.thaliana) to almost two thousand
base pairs (e.g. 1985 bp in the case of MIR156a
A.thaliana), however introns exceeding several
hundred bp in length are relatively rare. The ratios
of total intron length to the overall length of intron-
containing MIR gene range from around 10% to
almost 80%.
1.2 MIR gene transcription
Detailed characterization of a few
Arabidopsis miRNA precursors (pri-miR163, pri-
miR164a-c, pri-miR171a, pri-miR172b, pri-miR319a,
pri-miR824) showed presence of a 5' cap structure
and/or a 3' poly(A) tail (8-14). In addition, large-
scale analysis revealed that transcripts of 52 out
of 99 A.thaliana MIR loci studied undergo both
cap structure formation and polyadenylation (5).
Figure 1 Logos of intron sequences identified within A.thaliana MIR genes (A) 5' splice site (B) 3' splice
site. Logos were generated based on a multiple alignment of 23 sequences from 14 intron-containing MIR
transcripts using WebLogo v. 2.8.2. The overall height of each stack of symbols indicates the sequence
conservation at a given position, whereas the height of symbols within the stack indicates the relative
frequency of a nucleotide. Negative numbers refer to positions of nucleotides within exon, whereas
positive numbers denote intronic sequence.
3Plant MIR gene structures
It has been also shown that sequences matching
MIR loci from different plant species can be found
within respective EST (Expressed Sequence Tag)
libraries (15), suggesting that plant MIR gene
transcripts are commonly polyadenylated.
It is known that RNA Pol II-specific CTD
(carboxy-terminal domain) functions as a platform
for co-transcriptional recruitment of factors
involved in pre-mRNA processing including 5' end
cap formation and 3' end cleavage followed by
polyadenylation (reviewed in 16). Thereby
presence of both cap structures and poly(A) tails
in plant MIR gene transcripts provide indirect
evidence for the involvement of RNA Pol II in
their synthesis. Moreover, biocomputational
analysis has revealed that the vast majority of
the characterized plant MIR genes contain TATA
box-like sequence within core promoter and that
usually the first transcribed nucleotide is
adenosine preceded by a pyrimidine (5,7). These
data are consistent with RNA Pol II transcription
characteristics (17). Altogether it seems that RNA
Pol II is involved in generation of the majority of
plant pri-miRNAs, nevertheless it cannot be
excluded that similarly to animals transcription of
some of the plant MIR genes depends on RNA
Pol III catalytic activity (18).
1.3 Splicing of MIR primary transcripts
Studies on plant MIR gene exon/intron
structure revealed that the presence of introns
within their sequences is more rule than the
exception. Moreover, it has been found that some
of the pri-miRNAs encoded by intron-containing
MIR genes may undergo alternative splicing such
as exon skipping as well as alternative 5' and 3'
splice site selection (6). However as all identified
pri-miRNA isoforms contain miRNA sequence, the
functional meaning of this process remains
elusive.
It has been shown for protein-coding genes
that there is a direct connection between gene
transcription and further, co- or
posttranscriptional, processing of nascent pre-
mRNAs (subsection 1.2). It is also known that
alike pre-mRNAs plant MIR gene primary
transcripts commonly undergo further processing
including cap structure formation and
polyadenylation. Thus, it seems very likely that
also in terms of splicing both groups of RNA Pol
II transcripts show significant similarity. This
notion is further supported by the fact that based
on nucleotide sequence of 5' and 3' splice sites all
introns identified within MIR genes were
classified as U2-dependent type.
Interestingly, it has been revealed that
proteins involved in pre-mRNA splicing and
miRNA maturation show functional overlap
(subsection 1.4). Moreover, it has been shown
that assembly of miRNA maturation aparatus may
be initiated even before pri-miRNA splicing takes
place. Therefore one can assume that there is a
spatio-temporal co-occurence of pri-miRNA
splicing and the first steps of their DCL1-
dependent maturation. However, specific features
of MIR genes and/or MIR gene primary transcripts
determining the recruitment of proteins specific
for miRNA maturation process are yet to be
discovered.
1.4 MicroRNA primary precursor maturation
MicroRNAs (miRNAs) arise from MIR gene
transcripts maturated in a multistage process. The
core of plant miRNA primary precursors (pri-
miRNA) maturation machinery is formed by the
endonuclease DCL1 (DICER LIKE 1). DCL1-
dependent consecutive trimming of 5’ and 3’ ends
of miRNA precursors gives rise to the so-called
pre-miRNAs and, subsequently, miRNA:miRNA*
duplexes (9,19). DCL1 cooparates with at least
4 Szarzynska B. et al
five additional proteins: DAWDLE (DDL),
SERRATE (SE), CAP-BINDING PROTEIN 20
(CBP20) and CAP-BINDING PROTEIN 80/ABA
HYPERSENSITIVE 1 (CBP80/ABH1) forming
CAP-BINDING COMPLEX (CBC) and
HYPONASTIC LEAVES 1 (HYL1) also known as
DOUBLE STRANDED RNA BINDING PROTEIN
1 (DRB1) (reviewed in 20). DDL is a RNA-binding
protein and apart form pri-miRNA maturation it is
involved in biogenesis of endogenous siRNAs
(21). DDL has been shown to interact with DCL1
and is thought to guide DCL1 to miRNA
precursors. SE protein is crucial for the
accumulation of multiple miRNAs and trans-
acting small interfering RNAs (ta-siRNAs) and is
found in the SmD3/SmB nuclear bodies (D-
bodies) together with DCL1 and HYL1 (13,22). It
has been shown that SE and CBC are involved in
miRNA biogenesis pathway at the pri-miRNA
maturation step and that they co-operate in this
process (23,24). Moreover, results obtained by
Laubinger et al. (24) indicate the influence of both
SE and CBC on splicing of numerous pre-mRNAs
with more than 50% of transcripts in common.
According to the authors there is no evidence for
a particular requirement of the CBC and SE in
processing of pri-miRNAs encoded by intron-
containing MIR genes. Therefore the effects on
pre-mRNA splicing and miRNA processing may
reflect independent roles of the CBC and SE in
these two processes. Nevertheless, the impact of
cbp mutations on splicing is not limited to pre-
mRNAs, but similar effect has been also shown
for pri-miRNAs (6).
The HYL1 protein was primarily reported to
influence efficiency and precision of pri-miRNA
cleavage (25,26). However it has been recently
found that HYL1 couples splicing of MIR gene
primary transcripts and DCL1-dependent pri-
miRNA maturation (Figure 2) (6). These results
suggest that the HYL1 protein accompanies pri-
miRNAs from the very early steps of their
maturation. It is very likely that HYL1 is
incorporated into pri-miRNA maturation aparatus
immediately after MIR gene transcription or even
co-transcriptionally.
2 Intragenic MIR loci
2.1 Organization of intragenic MIR loci
In plants miRNA-coding sequences are
found mainly within intergenic regions (5), though
there are some exceptions. In Physcomitrella
patens ~30% of sequences encoding pre-miRNAs
overlaps with the annotated protein-coding loci
and is localized in the same orientation as the
annotated genes (27). In Arabidopsis thaliana
only ~6% of known miRNAs originate from
intragenic regions (4,28). For example miR162,
miR842, miR844, miR850 and miR852 are embedded
within an intron of a gene with no annotated
function (Figure 3). In the case of five other
species (miR402, miR837, miR838, miR853 and
miR862) miRNA sequences overlap with introns
of protein-coding genes. Plant genes contaning
miRNAs within their intron sequences show great
diversity of exon/intron organization. Among them
there are genes containing single intron
(At1g20860, At2g23348, At5g13890), as well as
multi-intron genes (At1g01040 and At2g25170
with 20 and 30 introns, respectively). The miRNA-
containing intron length vary greatly ranging from
several hundred (259 bp in the case of At5g08185)
to over two thousand (2620 bp in the case of
At4g13495). Moreover, there seems to be no rule
regarding localization of miRNA-containing
intron when imposed to the overall gene structure,
as there are examples of miRNA sequences
identified both within introns of UTR (in the case
of At2g23348 within 5' UTR and in At5g13890
within 3' UTR, respectively) and in introns located
within an ORF (At1g01040, At1g18880,
At1g77230, At2g25170, At3g23325).
5Plant MIR gene structures
Figure 2 Analysis of pri-miRNA and pre-mRNA splicing in A. thaliana wild-type plants, hyl1 mutant and
cbp20xcbp80(abh1) double mutant by semiquantitative RT-PCR. Schematic representations of the analyzed
transcripts with their exon (box) and intron (line) organizations are shown on the right. Note that they are
not drawn to scale. (A) Both unspliced and spliced forms of pri-miRNAs accumulate in the hyl1 mutant in
comparison to the wild-type plants. (B) The influence of the hyl1 mutation on splicing seems to be
restricted to pri-miRNAs, whereas CBP inactivation may affect splicing of both pri-miRNA and pre-mRNA
(C) The amount of cDNA was standardized to the ACTIN2 (ACT2) expression level.
Figure 3 Arabidopsis miRNAs encoded by sequences localized within introns of (A-D) protein-coding
genes (E-H) genes of unknown function (based on 4). At5g08185 gene containing pre-miR162a coding
sequence within the third intron and At1g01040 (DCL1) with pre-miR838 coding sequence localized
within the fourteenth intron are presented in the Figure 5. In the case of At2g25170 only the fragment
including exons 14-22 is shown. Genes are drawn to scale as given at the top of the picture. Black boxes
depict 5' and 3' UTRs, grey boxes - exons; black discontinuous lines - introns and black bold lines -
sequences coding for pre-miRNAs with their lengths given below. The overall lengths of gene transcripts
as well as gene functions are depicted on the left.
6 Szarzynska B. et al
Apart from the intronic miRNAs, the are
some examples of plant miRNAs embedded within
exon sequences. The entire A.thaliana pre-miR840
sequence is included within 3' UTR of At2g02750
transcript. At2g02750 is coding for a protein of
unknown function belonging to PPR
(pentatricopeptide repeat-containing) protein
family (29). Interestingly, At2g02750 sequence
partially overlaps WHY3 gene (WHIRLY3,
At2g02740) encoding miR840 predicted target
transcript, though MIR840 and WHY3 are
transcribed from the opposite strands. WHY3
encodes a homolog of potato p24, a DNA-binding
protein directed to plastids and functioning as a
transcriptional regulator of disease-resistance
genes. Mature miR840 sequence is complementary
to 3' UTR of WHY3 mRNA and can possibly direct
its cleavage. In Zea mays 18 miRNA-coding
sequences localized within exons of predicted
protein coding genes have been recently identified
(7). However, putative protein-coding genes in
which miRNAs have been found encode relatively
small proteins (of less than 120 aa) with no
conserved domains, therefore it seems plausible
that they are missannotated.
2.2 Trancription and splicing of intragenic
MIR loci
It has been shown that human intronic
miRNAs are usually coordinately expressed with
their host gene, suggesting that MIR and a host
gene undergo transcription under control of a
common promoter (30). However in plants little is
known regarding possible correlation between
host gene transcription and miRNA precursor
synthesis as well as their further processing. In
the case of intronic miRNAs a crosstalk between
assembly of spliceosome and miRNA biogenesis
machinery may significantly influence maturation
of both miRNA and a host mRNA. Three models
of intron-derived plant miRNA maturation have
been proposed (Figure 4): (A) DCL1-mediated
cleavage of the miRNA precursor from pre-mRNA
Figure 4 Working models of the intronic plant miRNA processing (based on 4). (A) Splicing of the pre-
mRNA and miRNA precursor cleavage are mutually exclusive and result in either functional mRNA or
miRNA formation. (B) Cleavage catalyzed by DCL1 occurs after spliceosom assembly allowing for
production of both miRNA and spliced host mRNA. (C) Cleavage of the miRNA precursor by DCL1
occurs after splicing at the intron lariat or de-branched intron stage. Grey boxes refer to exons, black
discontinuous lines - introns and black bold lines - hairpin structures containing miRNA, respectively.
7Plant MIR gene structures
and pre-mRNA splicing are mutually exclusive,
(B) DCL1 action does not impair pre-mRNA
splicing as it occurs after assembly of spliceosomal
complexes tethering the exons flanking the spliced
intron or (C) miRNA precursor is released from the
excised and linearized intron (4).
Interestingly, miR853 is found within an
intron of the gene coding for 10-kDa subunit of
splicing factor SF3b (At3g23325). However, the
influence of miR853 maturation on SF3b mRNA
processing and as a consequence, SF3b synthesis
level and splicing is not known (4,31). In
Arabidopsis two examples of splicing of the
primary transcripts containing miRNA sequences
within their introns have been investigated in
further detail. Both intronic miRNAs, miR162a and
miR838, are involved in the negative feedback
regulation of miRNA maturation pathway.
Sequence coding for miR162a is located within
the second intron of At5g08185 (Figure 5A) (4).
Primary transcript of this gene undergoes
alternative splicing resulting in the formation of
five different isoforms (AS1 - AS5). Apart from
the unspliced transcript only two splicing forms
(AS1 and AS2) may give rise to mature miR162a.
One possibility is the excision of pre-miRNA from
the released intron sequence (AS1). According
to the other scenario, removal of a part of the
intron encompassing from the canonical 5' splice
site to an alternative one immediately upstream
of the pre-miR162a sequence generates isoform
with an intact miRNA precursor sequence (AS2),
which can be possibly cleaved by DCL1. The
function of the three other identified splice
variants remains unknown. However, localization
of the characterized alternative splice sites
indicates the potential competition between
splicing complex assembly and folding of the
miRNA-containing intron region into stem-loop
secondary structure. The removal of an intron
fragment defined by an alternative 5' splice site
located within the terminal loop of the miRNA-
containing hairpin and alternative 3' splice-site (3
nt downstream from the standard 3' splice site)
dissects miRNA precursor (AS3-AS5) and makes
its maturation impossible. On the contrary, the
use of the alternative 3' splice site localized next
to the base of pre-miRNA hairpin structure at its
5' side of the base of the stem-loop (AS2) suggests
that this event enables folding of miRNA
precursor. Based on these results it seems very
likely that splicing of a non-protein-coding
At5g08185 transcript is influenced by miRNA
precursor secondary structure formation and/or
the assembly of the miRNA maturation complex.
In contrast to miR162a localized in the
transcript of unknown function, miR838 is
comprised within intron XIV of the DCL1 pre-
mRNA (28). The analysis of DCL1 transcript using
RACE technique allowed to identify apart from
the intact mature DCL1 mRNA two additional
groups of products, i.e. one terminating at the 3'
end of the exon XIV and the second - a
heterogenous pool of fragments with the 5' end
of the longest one within intron XIV (32) (Figure
5B). These results imply that due to competition
between splicing and miRNA production DCL1
primary transcript may undergo either splicing
generating full-length DCL1 mRNA or processing
by DCL1 itself resulting in the production of
mature miRNA and truncated DCL1 mRNA. In
this way miR383 takes part in DCL1 autoregulatory
feedback loop.
The above examples indicate that splicing
events and miRNA maturation can be mutually
exclusive. On the contrary, it has been shown that
splicing is required to produce some of the rice
miRNAs as the removal of an intron brings
together partial miRNA sequences located in
different regions of a primary non-protein coding
transcript, thereby forming miRNA sequence (33).
8 Szarzynska B. et al
Figure 5 (A) Exon/intron organization of A.thaliana MIR162a gene (At5g08185), fragments of its primary
transcript and alternative splicing forms (AS1-AS5) (based on 4). The function of variants depicted as
AS3-AS5 is not known. The unspliced transript, spliced out intron sequence (AS1) and AS2 form contain
miR162a sequence and can be possibly recognized by miRNA maturation machinery. Barrels represent
exons with 5' and 3' UTR regions marked in black. Introns are depicted with decontinuous lines with the
exception of pre-miRNA sequence marked with solid line. Alternative splicing events (1-4) are marked with
dotted lines. (B) Exon/intron organization of A.thaliana DCL1 (At1g01040) gene primary transcript (based
on 43). Exon regions coding for conserved protein domains are highlighted dark grey. NLS - bipartial
Nuclear Localization Signal, DUF - Domain of Unknown Function, PAZ - Piwi Argonaute Zwille domain,
dsRBDs - two double-stranded RNA-binding domains. Further details can be found in the text.
9Plant MIR gene structures
3 Partially overlapping MIR genes
Apart from intragenic miRNAs possibly co-
expressed with their host genes, A.thaliana MIR
loci partially overlapping with protein-coding
gene sequences have been identified. For example,
it has been found that A.thaliana miR777-coding
sequence overlaps At1g70650 5' UTR, however
sequence encoding the whole pre-miR777 extends
beyond 5' boundary of the protein-coding gene
(28,31). As in many cases MIR gene boundaries
haven’t been established, it is possible that there
are more examples of such overlapping genes
hidden in plant genomes. The mechanisms
regulating expression of such MIR gene- protein-
coding gene tandems remains unknown.
4 Loci coding for multiple miRNAs
4.1 MicroRNA clusters
In contrast to animal genomes with miRNA-
coding sequences often identified in close
proximity to each other and possibly co-
transcribed as polycistronic RNAs (34-37), in
plants such organization of MIR loci does not
seem to be common (38-40). Physcomitrella patens
seems to be an exemption within plant kingdom
as approximately one fourth of its known miRNAs
is localized in close proximity to another MIR loci.
Therefore it is very likely that they are
cotranscribed to polycistronic precursors
containing two or three miRNA stem-loops each
(27). In Oryza sativa four clusters of miR395-
coding sequences were identified (41). Sequences
encoding seven members of the miR395 family
(miR395a-g) are localized in the same orientation
on chromosome 4 and separated by about 120
base pairs. Identification of O.sativa Expressed
Sequence Tag (EST) containing sequences of
three miRNAs: 395a, 395b and 395c provided
evidence supporting the hypothesis that these
miRNAs may derive from a single transcript (15,42)
(Table 1).
4.2 MicroRNAs originating from the same
pri-miRNA
Endonucleolytic pri-miRNA cleavage
reactions are catalyzed by RNase III activity of
DCL (DICER LIKE) family members. In Arabidopsis
four DCL proteins have been identified (45, 46).
DCL1 is the key miRNA biogenesis enzyme,
producing canonical 20-21 nucleotide long
miRNAs with the exception of A.thaliana miR163
Table 1 Examples of plant co-transcribed miRNA clusters (15, 42-44). Star (*) refers to miRNA passanger
molecule.
miRNA family miRNA cluster Plant species
miR166 166a-1, 166a-2 Medicago truncatula
166a, 166b Glycine max
166f, 166g Glycine max
miR169 169a, 169b Gossypium herbaceum
169b, 169d Glycine max
169c, 169g* Glycine max, Glycine soja
miR171 171a, 171d Glycine max
miR395 395a, 395b, 395c Oryza sativa
10 Szarzynska B. et al
which is 24-nt in length (9,19). Interestingly, it has
been shown that DCL3 is involved in biogenesis
of 23-25 nucleotide long miRNAs generated from
the same miRNA precursors as the canonical ones
(47). It was found that conserved MIR genes
encode predominantly canonical miRNAs, whereas
recently evolved MIR genes mostly give rise to
both canonical and so called ‘long miRNAs’. One
can assume that in the case of pri-miRNAs encoded
by evolutionary young MIR genes there is a
competition between DCL1 and DCL3 and that
biogenesis of canonical and ‘long miRNAs’
deriving from the same precursor is mutually
exclusive. Interestingly, in A.thaliana the
expression level of DCL3 within inflorescence is
~10-fold higher when compared to leaves,
indicating possible preference for DCL3-dependent
pri-miRNAs maturation within this tissue.
ACKNOWLEDGMENTS:
This work was supported by two grants from
the Ministry of Higher Education and Sciences
of Poland - 3011/B/P01/2009/37 and NN301/03/
58/39, and a grant for scientific research from the
Dean of Biology Faculty, Adam Mickiewicz
University, Poznan, Poland (to LS) PBWB-8/2010
LITERATURE
1. Reddy ASN (2007) Alternative splicing of pre-
messenger RNAs in plants in the genomic
era. Annu Rev Plant Biol 58, 267-294.
2. Schnable PS, Ware D, Fulton RS, Stein JC,
Wei F, et al. (2009) The B73 maize genome:
complexity, diversity and dynamics. Science
326, 1112-1115.
3. Rodriguez A, Griffiths-Jones S, Ashurst JL,
Bradley A (2004) Identification of mammalian
microRNA host genes and transcription
units. Genome Res 14, 1902-1910.
4. Brown JWS, Marshall DF, Echeverria M
(2008) Intronic noncoding RNAs and
splicing. Trends Plant Sci 13, 335-342.
5. Xie Z, Allen E, Fahlgren N, Calamar A, Givan
SA, Carrington JC (2005) Expression of
Arabidopsis MIRNA genes. Plant Physiol
138, 2145-2154.
6. Szarzynska B, Sobkowiak L, Pant
BD,
Balazadeh S, Scheible
W-R, Mueller-Roeber
B, Jarmolowski,A,
Szweykowska-Kulinska
Z (2009) Gene structures and processing
of Arabidopsis thaliana HYL1-dependent
pri-miRNAs. Nucleic Acids Res 9, 3083–
3093.
7. Zhang L, Chia J-M, Kumari S, Stein J-C, Liu
Z, Narechania A, Maher ChA, Guill K,
McMullen MD, Ware D (2009) A genome-
wide characterization of microRNA genes
in maize. PLoS Genet 5, e1000716.
8. Aukerman MJ, Sakai H (2003) Regulation of
flowering time and floral organ identity by a
microRNA and its APETALA-like target
genes. Plant Cell 15, 2730-2741.
9. Kurihara Y, Watanabe Y (2004) Arabidopsis
microRNA biogenesis through Dicer-like 1
protein functions. Proc Natl Acad Sci USA
101, 12753-12758.
10. Baker CC, Sieber P, Wellmer F, Meyerowitz
EM (2005) The early extra petals1 mutant
uncovers a role for microRNA miR164c in
regulating petal number in Arabidopsis.
Curr Biol 15, 303-315.
11. Nicovics K, Blein T, Peaucelle A, Ishida T,
Morin H, Aida M, Laufs P (2006) The
balance between the MIR164A and CUC2
genes controls leaf margin serration in
Arabidopsis. Plant Cell 18, 2929-2945.
12. Kutter C, Schöb H, Stadler M, Meins Jr F,
Si-Ammour A (2007) MicroRNA-mediated
11Plant MIR gene structures
regulation of stomatal development in
Arabidopsis. Plant Cell 19, 2417-2429.
13. Song L, Han M-H, Lesicka J, Fedoroff N
(2007) Arabidopsis primary microRNA
processing proteins HYL1 and DCL1
define a nuclear body distinct from the
Cajal body. Proc Natl Acad Sci USA 104,
5437-5442.
14. Warthmann N, Das S, Lanz Ch, Weigel D
(2008) Comparative analysis of the MIR319a
microRNA locus in Arabidopsis and related
Brassicaceae Mol Biol Evol 25, 892-902.
15. Jones-Rhoades MW, Bartel DP (2004)
Computational identification of plant
microRNAs and their targets, including a
stress-induced miRNA. Moll Cell 14, 787-799.
16. Bentley DL (2005) Rules of engagement: co-
transcriptional recruitment of pre-mRNA
processing factors. Curr Opin Cell Biol 17,
251-256.
17. Shahmuradov IA, Gammerman AJ, Hancock
JM, Bramley PM, Solovyev VV (2003)
PlantProm: a database of plant promoter
sequences. Nucleic Acids Res 31, 114-117.
18. Borchert GM, Lanier W, Davidson BL (2006)
RNA polymerase III transcribes human
microRNAs. Nat Struct Mol Biol 13, 1097-
1101.
19. Park W, Li J, Song R, Messing J, Chen X
(2002) CARPEL FACTORY, a Dicer homolog,
and HEN1, a novel protein, act in microRNA
metabolism in Arabidopsis thaliana. Curr
Biol 12, 1484-1495.
20. Voinnet O (2009) Origin, biogenesis, and
activity of plant microRNAs. Cell 136, 669-
687.
21. Yu B, Bi L, Zheng B, Ji L, Chevalier D,
Agarwal M, Ramachandran V, Li W,
Lagrange T, Walker JC, Chen X (2008) The
FHA domain proteins DAWDLE in
Arabidopsis and SNIP1 in humans act in
small RNA biogenesis. Proc Natl Acad Sci
USA 105, 10073-10078.
22. Fujioka Y, Utsumi M, Ohba Y, Watanabe Y
(2007) Location of a possible miRNA
processing site in SmD3/SmB nuclear bodies
in Arabidopsis. Plant Cell Physiol 48, 1243-
1253.
23. Kim S, Yang J-Y, Jang I-Ch, Prigge MJ, Chua
N-H (2008) Two CAP BINDING PROTEINS
CBP20 and CBP80 are involved in
processing primary miRNAs. Plant Cell
Physiol 11, 1634-1644.
24. Laubinger S, Sachsenberg T, Zeller G, Busch
W, Lohmann JU, Raetsch G, Weigel D (2008)
Dual roles of the nuclear cap-binding
complex and SERRATE in pre-mRNA
splicing and microRNA processing in
Arabidopsis thaliana. Proc Natl Acad Sci
USA 105, 8795-8800.
25. Kurihara Y, Takashi Y, Watanabe Y (2006)
The interaction between DCL1 and HYL1 is
important for efficient and precise
processing of pri-miRNA in plant microRNA
biogenesis. RNA 12, 206-212.
26. Dong Z, Han MH, Fedoroff N (2008) The
RNA-binding proteins HYL1 and SE
promote in vitro processing of pri-miRNA
by DCL1. Proc Natl Acad Sci USA 105, 9851-
9852.
27. Axtell MJ, Snyder JA, Bartel DP (2007)
Common functions for diverse small
RNAs in land plants. Plant Cell 19, 1790-
1789.
28. Rajagopalan R, Vaucheret H, Trejo J, Bartel
DP (2006) A diverse and evolutionarily fluid
set of microRNAs in Arabidopsis thaliana.
Genes Dev 20, 3407-3425.
12 Szarzynska B. et al
29. The Arabidopsis Information Resource
TAIR (http://www.arabidopsis.org/).
30. Baskerville S, Bartel DP (2005) Microarray
profiling of microRNAs reveals frequent
coexpression with neighboring miRNAs
and host genes. RNA 11, 241-247.
31. Map Viewer (NCBI, http://
www.ncbi.nlm.nih.gov/projects/mapview/
static/MapViewerHelp.html)
32. Xie Z, Kasschau KD, Carrington JC (2003)
Negative feedback regulation of Dicer-
Like1 in Arabidopsis by microRNA-guided
mRNA degradation. Curr Biol 13, 784-789.
33. Sunkar R, Zhu J-K (2005) Cloning and
characterisation of microRNAs from rice.
Plant Cell 17, 1397-1411.
34. Lagos-Quintana M, Rauhut R, Lendeckel W,
Tuschl T (2001) Identification of novel genes
coding for small expressed RNAs. Science
294, 853-858.
35. Lau NC, Lim LP, Weinstein EG, Bartel DP
(2001) An abundant class of tiny RNAs with
probable regulatory roles in Caenorhabditis
elegans. Science 294, 858-862.
36. Lee Y, Jeon K, Lee J-T, Kim S, Kim VN (2002)
MicroRNA maturation: stepwise processing
and subcellular localization. EMBO J 21,
4663-4670.
37. Aravin AA, Lagos-Quintana M, Yalcin A,
Zavolan M, Marks D, Snyder B, Gaasterland
T, Meyer J, Tuschl T (2003) The small RNA
profile during Drosophila melanogaster
development. Dev Cell 5, 337-350.
38. Talmor-Neiman M, Stav R, Frank W, Voss B,
Arazi T (2006) Novel micro-RNAs and
intermediates of micro-RNA biogenesis from
moss. Plant J 47, 25-37.
39. Chuck G, Cigan MA, Saeteurn K, Hake S
(2007) The heterochronic maize mutant
Corngrass1 results from overexpression of
a tandem microRNA. Nat Genet 39, 544-549.
40. Zhao T, Li G, Mi S, Li S, Hannon GJ, Wang
XJ, Qi Y (2007) A complex system of small
RNAs in the unicellular green alga
Chlamydomonas reinhardtii. Genes Dev 21,
1190-1203.
41. miRBase::Sequence Database http://
www.mirbase.org/; Griffiths-Jones S, Saini
HK, van Dongen S, Enright AJ (2008)
miRBase: tools for microRNA genomics.
Nucl Acids Res 36, D154-D158.
42. Zhang B, Pan X, Cannon ChH, Cobb GP,
Anderson TA (2006) Conservation and
divergence of plant microRNA genes. Plant
J 46, 243-259.
43. Boualem A, Laporte P, Jovanovic M, Laffont
C, Plet J, Combier JP, Niebel A, Crespi M,
Frugier F (2008) MicroRNA166 controls root
and nodule development in Medicago
truncatula. Plant J 54, 876-887
44. Zhang B, Pan X, Stellwag EJ (2008)
Identification of soybean microRNAs and
their targets. Planta 229, 161-182
45. Golden TA, Schauer SE, Lang JD, Pien, S
Mushegian AR, Grossniklaus U, Meinke
DW, Ray A (2002) SHORT
INTEGUMENTS1/SUSPENSOR1/CARPEL
FACTORY, a Dicer homolog, is a maternal
effect gene required for embryo development
in Arabidopsis. Plant Physiol 130, 808-822.
46. Schauer SE, Jacobsen SE, Meinke DW, Ray
A (2002) DICER-LIKE1: Blind men and
elephants in Arabidopsis development.
Trends Plant Sci 7, 487-491.
47. Vazquez F, Blevins T, Ailhas J, Boller T,
Meins Jr F (2008) Evolution of
Arabidopsis MIR genes generates novel
microRNA classes. Nucleic Acids Res 36,
6429-6438.
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