gene structures and processing of plant pri-mirnas

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


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).


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.


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.


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).


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.


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


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


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.


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.

gene structures and processing of plant pri-mirnas

Documents