Báo cáo khoa học: Genome-wide analysis of clustering patterns and flanking characteristics for plant microRNA genes doc

Analysis of the promoter architecture of different miRNA genes in plants revealed significant differences in the number and distribution of core promoters between intergenic miRNAs and in

characteristics for plant microRNA genes

Meng Zhou1,*, Jie Sun1,*, Qiang-Hu Wang1,*, Li-Qun Song2, Guang Zhao1, Hong-Zhi Wang2, Hai-Xiu Yang1and Xia Li1

1 College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China

2 Department of Internal Medicine, Affiliated Hospital of Heilongjiang University of Chinese Medicine, Harbin, China

Introduction

MicroRNAs (miRNAs), 21–24 nucleotides in

length, are a large class of endogenous, noncoding

small RNA molecules that regulate gene expression

at the post-transcriptional level in animals and plants

[1–4] The first microRNA – lin-4 – was discovered

in 1993 in Caenorhabditis elegans through forward

genetic screens [5] The first plant miRNA was

dis-covered in Arabidopsis thaliana in 2002 [6,7] Plant

miRNA genes are mostly transcribed into primary

miRNA transcripts (pri-miRNAs) by RNA

polymer-ase II (Pol II) The pri-miRNAs are processed by DICER-LIKE 1 (DCL1) into stem–loop pre-miRNAs

in the nucleus Then, pre-miRNAs are processed by DCL1 in the nucleus and exported to the cytoplasm, possibly through the action of the plant exportin 5 or-thologue HASTY and other unknown factors Mature RNA duplexes excised from pre-miRNAs

(miR-NA⁄ miRNA*, where miRNA is the guide strand and miRNA* is the degraded strand) are methylated by HEN1 The guide miRNA strand is then incorporated

Keywords

clustering patterns; flanking regions; motif;

plant microRNA gene; sequence

characteristics

Correspondence

Xia Li, College of Bioinformatics Science

and Technology, Harbin Medical University,

Harbin 150081, China.

Fax: +86 045186615922

Tel: +86 045186669617

E-mail: lixia@hrbmu.edu.cn

*These authors contributed equally to this

work

(Received 11 October 2010, revised 7

December 2010, accepted 7 January 2011)

doi:10.1111/j.1742-4658.2011.08008.x

MicroRNAs (miRNAs) have been proven to play important roles at the post-transcriptional level in animals and plants To investigate clustering patterns and specific sequence characteristics in the flanking regions of plant miRNA genes, we performed genome-wide analyses of Arabidopsis thaliana, Populus trichocarpa, Oryza sativa and Sorghum bicolor Our results showed that miRNA pair distances were significantly higher than would have been expected to occur at random and that the number of miRNA gene pairs separated by very short distances of < 1 kb was higher than of protein-coding gene pairs Analysis of the promoter architecture of different miRNA genes in plants revealed significant differences in the number and distribution of core promoters between intergenic miRNAs and intragenic miRNAs, and between highly conserved miRNAs and low conserved or nonconserved miRNAs We applied two motif-finding algo-rithms to search for over-represented, statistically significant sequence motifs, and discovered six species-specific motifs across the four plant spe-cies studied Moreover, we also identified, for the first time, several signifi-cantly over-represented motifs that were associated with conserved miRNAs, and these motifs may be useful for understanding the mechanism

of origin of new plant miRNAs The results presented provide a new insight into the transcriptional regulation and processing of plant miRNAs

Abbreviations

miRNA, microRNA; Pol II, RNA polymerase II; pri-miRNAs, primary miRNA transcripts; TSSs, transcription start sites.

into AGO proteins to carry out the silencing reactions

[1,2]

In plants, Xie et al [8] identified transcription start

sites (TSSs) for 63 miRNA primary transcripts in

A thaliana and found the TATA box motif in their

core promoter regions Unlike animal miRNAs, the

vast majority of plant miRNAs are intergenic but not

intronic [2,9] Several studies have characterized the

upstream sequences of intergenic miRNAs in model

organisms and found the same type of promoters as in

the protein-coding genes of most of the intergenic

miRNAs [10–12] Furthermore, Zhou et al [11] also

discovered some interesting sequence motifs that are

specific to intergenic miRNAs in four different model

species For all other miRNAs located within the

introns of protein-coding genes, little is known about

their transcriptional regulatory element These

intra-genic miRNAs are possibly transcribed with, or

inde-pendently of, the host genes Recently, Heikkinen

et al [13] examined the upstream sequences of

miR-NAs in C elegans and Caenorhabditis briggsae, and

discovered a sequence motif – GANNNNGA –

com-mon to all miRNAs, including intragenic miRNAs In

rice (Oryza sativa), some intragenic miRNAs were

found to contain class II promoters in upstream

sequences [10] However, the complex transcriptional

regulation mechanisms of plant miRNAs still remain

largely unknown

Although many efforts have been directed towards

examining clustering patterns and the sequence

charac-teristics of the upstream sequences of miRNA genes in

animals in an attempt to understand transcriptional

regulation [11,13–16], similar analyses have been

per-formed only for a relatively small number of miRNAs

in plants, and these were limited to A thaliana and

O sativa Recently, increasing numbers of plant

miRNAs have been identified through forward

genet-ics, direct cloning and computational prediction An

increasing number of plant miRNAs provide a good

opportunity to uncover complex transcriptional

regu-lation mechanisms for plant miRNAs In our study,

we performed computational approaches, based on

genome-wide analyses, to examine the clustering

pat-terns of plant miRNAs In addition, we analyzed

regions, up to 2 kb upstream and up to 1 kb

down-stream, of miRNA stem–loop sequences in four plant

species, to identify characteristic sequence motifs We

hope that the present results can improve the current

understanding of transcriptional regulation and

pro-cessing of plant miRNAs and provide useful knowledge

for understanding the mechanism of the origin and

computational identification of new miRNAs in plants

Results and Discussion Analysis of clustering patterns of miRNA genes

in four plant genomes Many previous studies have shown that miRNA genes tend to be present as clusters within a region of several kilobases in animal genomes [17–20] In contrast, plant miRNA genes are rarely arranged in tandem [1] To further explore the clustering patterns of miRNAs in plant genomes, we computed the distances between same-strand consecutive miRNA genes of four plant species to analyze the distance distribution of miRNA genes in different plant species based on reported miR-Base coordinates The cumulative distance distribution

of the miRNA gene pairs is presented in Fig 1 and shows that 17.71%, 26.94% and 29.07% of the

miR-NA gene pairs are separated by regions of < 1, 10 and 100 kb, respectively, which are much smaller than the regions separating animal miRNA gene pairs Fur-thermore, we compared the distance distribution of the miRNA gene pairs with the distance distribution of protein-coding genes in four plant genomes (Fig 1)

We found that more miRNA gene pairs than protein-coding gene pairs were separated by very short dis-tances of < 1 kb To evaluate the statistical signifi-cance of the clustering patterns of miRNA genes in the four plant species studied, we also compared the distances of the miRNA gene pairs with random dis-tances, as described in the Materials and methods, and found that the miRNA gene pair distances were statis-tically significantly higher than expected at random (P < 0.001) To identify more characteristics of

miR-NA clusters in plant genomes, we defined 10 kb as the maximum inter-miRNA distance for two miRNA genes to be considered as clustered because 26.94% of the miRNA gene-pair distances were < 10 kb and extending the threshold to 100 kb added relatively few miRNA gene pairs Furthermore, the relatively small distance prevented overestimation of the number of clusters and made our analysis more stringent Accord-ing to this definition, we examined the characteristics

of potential clusters within maximum inter-miRNA distance of 2-,5- and 10-kb (Table 1) Our study revealed that the number of members in miRNA clus-ters at very short gene-pair distances in O sativa and Sorghum bicolorwas significantly larger than in A tha-liana and Populus trichocarpa (P < 0.01; two-sample t-test) This may suggest that miRNA clusters in monocots are larger than those in eudicots This spe-cific clustering pattern of miRNAs may be indicative

of functional divergence of the miRNA cluster in

miRNA-mediated gene regulation between monocots

and eudicots Furthermore, miRNA clusters in plants

are frequently found to have smaller size of cluster

compared with miRNA clusters in animals (P < 0.01;

two-sample t-test) In animals, a large proportion of

known miRNAs are arranged in clusters For example,

48% of human miRNAs appear as clusters within a

maximum inter-miRNA distance of 10 kb [21] and

50% of miRNAs appear as clusters within a maximum

inter-miRNA distance of 3 kb in the zebrafish genome

[22] In contrast to patterns of clustering found in

ani-mal miRNAs, only a sani-mall proportion of plant

miR-NAs (25.35% in A thaliana, 17.09% in P trichocarpa,

22.29% in O sativa and 21.62% in S bicolor) were

found to be clustered within a 10-kb region in our

study It has been demonstrated that miRNA families

are preferentially expressed in eudicots relative to

monocots [23] Our analysis further indicated that

most plant miRNA clusters are composed of family

members and are located in intergenic regions, which

is consistent with previous studies in plants [10,24,25]

Our results imply that the size of the miRNA cluster

may contribute to preferential expression in eudicots

relative to monocots Li et al [25] suggested that the

co-transcription of similar or identical miRNAs in

clusters for plants may be involved in gene dosage effect

Analysis of the core promoter of the class II promoter in plant miRNA genes

miRNA genes were determined to be part of the poly-cistronic transcript if the pairwise distance of two miRNAs on the same chromosome was < 10 kb For miRNAs in polycistronic transcripts, only sequences upstream of the 5¢ pre-miRNAs and downstream of the 3¢ pre-miRNAs were chosen to represent the poly-cistronic transcript As described in the Materials and methods, we used the TSSP-TCM program to initially search for the putative core promoter of the class II promoter occurring in 2-kb upstream sequences of miRNAs in the four plant species studied We identi-fied 130 (77.8%) miRNAs in A thaliana, 145 (89%) miRNAs in P trichocarpa, 233 (71.5%) miRNAs in

O sativa and 102 (81.6%) miRNAs in S bicolor to contain the core promoter of the class II promoter, suggesting that a significant proportion of plant miRNA genes have resident Pol II promoters in upstream regions It is generally accepted that miRNA genes located in the intronic regions as part of the host

Fig 1 Cumulative distance distribution of miRNA genes and protein-coding genes in four plant species The neighbour distances between every two same-strand miRNA genes or protein-coding genes in the same chromosome were calculated The distance is drawn on a loga-rithmic scale.

gene are expressed from the host gene promoters [26,27] However, a recent study on intergenic⁄ intronic and conserved⁄ nonconserved miRNA genes in rice revealed that several intronic miRNA genes in rice have a class II promoter, and rice miRNAs with more than one promoter appear to be conserved [10], thus implying that different sequence characteristics may be presented in upstream regions of different miRNA genes in plants To further explore the promoter archi-tecture of different miRNAs in plants and the relation-ship between the number of Pol II promoters and the degree of conservation of miRNAs, we classified four plant miRNA genes into two types (intergenic miR-NAs and intragenic miRmiR-NAs) based on their genomic locations Then, the miRNAs from the four plant spe-cies studied were divided into three groups (based on evolutionary conservation across all plant species, as described in the Materials and methods): highly con-served miRNAs, low concon-served miRNAs and noncon-served miRNAs The results are summarized in Fig 2

As shown in Fig 2A, we found a significant difference between intergenic miRNAs and intragenic miRNAs

in the numbers of class II promoters in the upstream regions (P < 0.001; two-sample t-test) The miRNAs lying between protein-coding genes usually contained more class II promoters in their upstream sequences (on average 1.4 per miRNA) than those miRNAs lying within the introns (on average 0.7 per miRNA) in the four plant species studied These results strongly indi-cate that most intergenic miRNAs are transcribed by RNA polymerase II in plants, and provide additional evidence that a significant proportion of intragenic miRNAs have Pol II promoters It suggests that these intragenic miRNAs may be transcribed as an indepen-dent unit from their own promoter However, in plants, a small number of miRNAs with no class II promoter may be transcribed through other transcrip-tional mechanisms, such as the host gene promoter Further studies carried out to explore whether there is

a relationship between the number of Pol II promoters and the degree of miRNA conservation revealed that the number of Pol II promoters in the upstream sequences of highly conserved miRNAs was signifi-cantly higher than in low conserved (P < 0.001; sample t-test) and in nonconserved (P < 0.001; two-sample t-test) miRNAs As shown in Fig 2B, only 13.67% of highly conserved miRNAs had no Pol II promoter, which is significantly lower than in low con-served miRNAs (31.14%) (P < 0.01; Fisher’s exact test) and in nonconserved miRNAs (26.76%) (P < 0.05; Fisher’s exact test) On the contrary, 50.13% of highly conserved miRNAs have at least two Pol II promoters, whereas only 27.38% of low

f The

served miRNAs (P < 0.001; Fisher’s exact test) and

23.94% of nonconserved miRNAs (P < 0.0001;

Fish-er’s exact test) have at least two Pol II promoters

However, there was no significant difference in the

number of Pol II promoters in upstream sequences

between low conserved and nonconserved miRNAs in

plants Taken together with the findings of the study

performed by Cui et al [10], our results provide a

more comprehensive understanding of the relationship

between the number of Pol II promoters and the

degree of miRNA conservation in plant genomes

Highly conserved miRNAs may be associated with

more Pol II promoters (on average 1.72 per miRNA)

than low conserved and nonconserved miRNAs (on average 1.13 and 1.05 per miRNA, respectively) in plants It has been demonstrated that the highly con-served miRNAs are likely to be central regulators and are highly expressed [28,29] The results of one study suggested that less conserved miRNAs rarely had obvi-ous effects on plant morphology [30] Therefore, we speculate that the increased number of Pol II promot-ers located in the upstream regions of highly conserved miRNAs may have an important effect on the high levels of expression of highly conserved miRNAs

To further characterize the putative core promoter

of the Pol II promoter in the upstream sequences of

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Intergenic

Intragenic Intergenic Intragenic

Intergenic

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The number of core promoter The number of core promoter

The number of core promoter

0 1 2 ≥3

The number of core promoter

The number of core promoter 100%

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Fig 2 Distribution of miRNA genes with

the same number of putative core

promot-ers (A) The percentage of miRNA genes

occurring between protein-coding genes or

within the introns in four plant species.

(B) The percentage of miRNA genes with

different degrees of conservation.

plant miRNAs, we examined the distribution of the

putative core promoter in 2-kb upstream regions of

miRNAs in the four species of plant studied In these

four plant species, the vast majority of the predicted

core promoters of the Pol II promoters were found to

lie within a 900-bp region upstream of the miRNAs

Distribution analysis of core promoter localization in

2-kb regions upstream of the miRNAs from the four

species of plants studied showed that 50.4% of the

putative core promoters of the Pol II promoter were

located within 0–1 kb, 26.8% were located within 1–

1.5 kb and 22.8% were located within 1.5–2 kb,

respectively of the miRNA A recent study on rice

(O sativa) suggested that the majority of TSSs and

TATA-boxes are found within 0–400 bp upstream of

the miRNA [10] Here, we found a similar distribution

of the putative core promoter in upstream regions of

miRNAs in four plant species As shown in Fig 3A, a

significant number of putative core promoters of the Pol II promoter were found to be located within the 400-bp upstream regions in three plant species, although the putative promoters in O sativa were dis-tributed mainly from 0 to 0.4 kb and from 1.6 to 2 kb Together, these results indicate that this distribution pattern of putative core promoters seems to be con-served in the 2-kb region upstream of miRNAs in dif-ferent plant species, and provide additional evidence that the core promoter regions of most miRNAs are close to pre-miRNA hairpins in plants Fig 3B shows the distribution of the core promoter in upstream sequences in view of the evolutionary conservation of plant miRNAs We found that the distribution pattern

of the core promoter in upstream regions was different between highly conserved miRNAs and low conserved

or nonconserved miRNAs Highly conserved miRNAs tend to contain more core promoters within the

400-bp region upstream of the miRNA However, core promoters are distributed mainly in the 0 to )0.4 kb, )0.8 to )1.2 kb and )1.6 to )2 kb regions upstream

of low conserved miRNAs, and, in contrast, core pro-moters are evenly distributed in upstream regions of nonconserved miRNAs These results suggest that there is a relationship between the distribution pattern

of core promoters and the degree of miRNA conserva-tion in plants Based on these observaconserva-tions, we pro-pose that the core promoter of Pol II promoters in the close proximal promoter region of miRNAs may play

a more effective, or even a greater, role for efficient transcription initiation

Analysis of specific sequence motifs in four plant species

To further identify specific characteristic motifs in the flanking regions of miRNAs in four plant species, we performed motif analysis to search for over-repre-sented and statistically significant motifs in the flank-ing regions up to 2 kb upstream and 1 kb downstream from the miRNA stem–loop sequences First of all, we used RepeatMasker with default settings to mask repeats in all upstream and downstream sequences, and then used two motif-finding tools – MEME and MotifSampler – to identify over-represented motifs Finally, we carried out whole-genome Monte Carlo simulation analysis to assess the specificity and signifi-cance of motifs identified, as described in the Materials and methods Motifs whose Z-scores were > 2.0 were considered as over-represented and statistically signifi-cant motifs Several signifisignifi-cantly over-represented spe-cies-specific motifs were identified in the flanking regions of four plant species All the species-specific

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–0.4 kb –0.8 kb –1.2 kb –1.6 kb –2 kb

–0.4 kb –0.8 kb –1.2 kb –1.6 kb –2 kb

Highly conserved Low conserved Non-conserved

Fig 3 Histograms of distances between putative core promoters

and miRNA stem–loop sequences The horizontal axis shows the

positions of putative core promoters with respect to the

corre-sponding miRNA stem–loop sequences, and the vertical axis shows

the percentage of putative core promoters at the specified

posi-tions (A) Percentage of putative core promoters at the specified

positions in different plant species (B) Percentage of putative core

promoters at the specified positions for miRNAs with a different

degree of conservation.

motifs found in the four plant species studied are

shown in Table 2 The motif M2, represented by the

consensus sequence TTAGGGTTTC, has also been

found in A thaliana by Zhou et al [11] Moreover, we

also discovered a novel motif – M1 – with a Z-score

value of 10.62 that is specific to A thaliana In order

to gain a deeper insight into the function of these

spe-cies-specific motifs, we compared our spespe-cies-specific

motifs against known transcription factors in plants

from the PlantCARE database [31] Only one motif

(M5) was already a known transcription factor in

plant promoters We found that M5, with the

consen-sus sequence GCATGCATGC, is an RY cis-acting

regulatory element involved in seed-specific regulation

in both monocot and eudicot species of plants [32,33]

Although the functions of other species-specific motifs

are still unknown, we found that some motifs have

repeat sequences in their consensus M5 has two copies

of GCAT, and M3, which can be considered as GCA-repeats Palindromic patterns have been found in the binding sites of some transcription factors in plants and animals [34,35] In contrast to A thaliana,

P trichocarpa and S bicolor, we could not detect any significant species-specific motifs in the flanking regions of miRNAs in O sativa, although a previous study has identified three specific motifs in the promot-ers of miRNAs in O sativa [11] Our analysis suggests that these species-specific motifs are associated with different specific functions, and may play an important role in species-specific transcriptional regulation net-works of miRNA genes or contribute to the formation

of species-specific miRNAs in plants However, their functions need to be investigated in further studies Furthermore, these species-specific motifs will be useful

in the computational identification of species-specific miRNAs in plants

Table 2 Significantly over-represented species-specific sequence motifs identified in the flanking regions of the three plant species studied.

Consensus

a The consensus sequence represents a sequence of the most frequent base at each position b The motif logos show the information con-tent present at each position in the sequence c The expected frequencies of motifs in a random database of the same size d The Z-score value was obtained by whole-genome Monte Carlo simulation analysis.

The mechanism by which new plant miRNAs

origi-nate is not fully understood It is believed that the

ori-gin of new plant miRNAs is dependent on duplication

and inversion events [36–38] However, several lines of

evidence have also suggested that new plant miRNA

genes can arise from foldback sequences, which are

under the control of transcriptional regulatory

sequences [39,40] In order to determine whether some

significantly over-represented sequence motifs are

related to the degree of conservation of miRNA genes

in plants, we classified the miRNA genes of four plant

species into highly conserved miRNAs, low conserved

miRNAs and nonconserved miRNAs, as described in

the Materials and methods We then examined the

upstream sequences and downstream sequences of

these miRNA genes to reveal characteristic sequence

motifs Several significantly over-represented motifs

associated with the degree of miRNA conservation are

identified and listed in Table 3 Two motifs

respectively), which have repetitive and palindromic

patterns in their consensus sequences, were found to

be significantly over-represented in highly conserved

plant miRNAs and therefore these motifs can be

con-sidered as CATG repeats and CTAG repeats,

respec-tively However, we did not find any significantly

over-represented sequence motifs in the flanking sequences

of nonconserved miRNAs in the four plant species In

contrast to nonconserved miRNA genes that have a

single copy, conserved miRNA genes are usually

multi-copy [25] miRNAs that are highly conserved across

plant species must have originated a long time ago and

experienced many genome-duplication events It has

been shown that the duplication events for miRNA gene evolution in plants not only involve the region that is transcribed but also the miRNA promoter regions [41,42] This might indicate that these signifi-cantly over-represented sequence motifs in highly con-served and low concon-served miRNAs are evolutionarily related elements that play important functional roles in evolutionarily conserved regulatory systems in plants

or are associated with duplication events for miRNA gene evolution in plants, although the functionality of these computationally identified conserved motifs remains to be experimentally validated

Conclusions

In this study, we concentrated our efforts on clustering patterns and flanking characteristics that might be involved in the transcriptional regulation and process-ing of plant miRNAs, includprocess-ing the miRNAs located

in the intergenic area and in the protein-coding area whose possible sequence characteristics were not stud-ied earlier Previous studies have revealed that miR-NAs located in close genomic proximity to each other are co-transcribed as polycistronic units [24,43,44] Therefore, we performed genome-wide analysis to examine the clustering patterns of the miRNAs in four species of plant The pairwise distance analysis results

of same-strand consecutive miRNAs suggested that the distances between the four plant miRNAs are statisti-cally significantly higher than expected at random (P < 0.001) Comparison of the miRNA pair distances with the pair distances of protein-coding genes revealed that plant miRNAs are more clustered than

Table 3 Significantly over-represented sequence motifs related to the conservation of miRNAs.

a The consensus sequence represents a sequence of the most frequent base at each position b The motif logos show the information con-tent present at each position in the sequence c The expected frequencies of motifs in a random database of the same size d The Z-score value obtained by whole-genome Monte Carlo simulation analysis.

protein-coding genes in the very short pairwise

dis-tances of < 1 kb Then, we characterized the putative

core promoter of Pol II promoters in plant miRNA

upstream sequences Our results suggest that most

plant miRNAs contain the core promoter of Pol II

promoters that are close to pre-miRNA hairpins

Analysis of promoter architecture for different miRNA

genes in plants reveals significant differences in the

number and distribution of core promoters between

intergenic miRNAs and intragenic miRNAs, and

between highly conserved miRNAs and low or

non-conserved miRNAs We applied two motif-finding

tools to search for over-represented, statistically

signifi-cant sequence motifs in the flanking regions of

miRNAs in different plant species Six motifs were

found to be species-specific motifs in three plant

species and included some previously known

species-specific motifs and some novel species-species-specific motifs

We also identified three specific motifs associated with

the degree of miRNA conservation

Compared with previous studies, our study

system-atically explored clustering patterns and the

character-istics of flanking regions up to 2 kb upstream and 1 kb

downstream of miRNA stem–loop sequences, and

extended the results on a small number of miRNAs in

A thaliana and in O sativa to all known miRNAs in

four plant species It remains largely unknown whether

there are some motifs related to the degree of

conser-vation of miRNAs In order to dissect this question,

we classified the miRNA genes of the four plant

species studied into three groups, according to their

conservation, and examined characteristic sequence

motifs in the flanking sequences of these miRNA

genes Several significant motifs appeared to be related

to the degree of miRNA conservation We hope that

our results can contribute to gaining a better

under-standing of transcriptional regulation and

process-ing of miRNAs and provide useful data for further

computational identification of miRNAs in plants

Also, we anticipate that these motifs related to the

degree of miRNA conservation may be useful for

understanding the mechanism of the origin of new

plant miRNAs

Materials and methods Data sets

To obtain the upstream and downstream sequences of plant miRNA genes, we chose four species of plant (A thaliana,

P trichocarpa, O sativa and S bicolor) to study clustering patterns and sequence characteristics in the flanking regions

of plant miRNA genes because the number of miRNA genes in these four plant species is relatively large and the genome sequences are relatively complete All known miRNAs and genome coordinates in these four plant spe-cies were downloaded from the miRBase Sequence Data-base, release 16 (http://www.mirbase.org/) [45] The genome sequences and the protein-coding genes of A thaliana and

S bicolor were downloaded from MapViewer in National Center for Biotechnology Information (http://www.ncbi nlm.nih.gov/) The genome sequences of P trichocarpa and

O sativa and the protein-coding genes were downloaded from the Poplar site on Phytozome v6.0 (P trichocarpa v2.0) (http://www.phytozome.net/poplar) [23] and TIGR Oryza Pseudomolecules (version_6.0) [46], respectively Then, we extracted sequences up to 2 kb upstream and up

to 1 kb downstream from all available miRNA precursors

in the four plant species A detailed description of the data set used in our study is shown in Table 4

Conservation analysis of miRNA in the four plant species studied

To determine the degree of conservation of miRNA in the four plant species, we performed a sequence-based homol-ogy search for known miRNAs from the four plants to detect both closely related and distantly related homo-logues First, known miRNA hairpin sequences from the four plants were aligned against all known miRNA hairpin sequences in monocots and eudicots using standalone BLAST (blastn, version 2.2.27) The hairpin sequences were considered as homologues when they exhibited a minimum sequence identity of 85% over an alignment length of at least 90% Second, ClustalW [47] was used to compare mature miRNA sequences for a search of homologues We adopted mature miRNA sequences matching at least 18 nucleotides and left 0–3 nucleotides for possible sequence

Table 4 Detailed description of the data set in our study.

Species

Version of genome annotation

No of miRNAs

No of polycistronic transcripts

No of upstream sequences

No of downstream sequences

variations [19] Finally, we divided the miRNAs of the four

plant species into three groups: the miRNAs whose

homo-logues were found simultaneously in monocots and eudicots

were considered as highly conserved miRNAs; those found

only in monocots or eudicots were considered as low

con-served miRNAs; and those found only in one species were

considered as nonconserved miRNAs

Analysis of clustering patterns

To study the clustering patterns of miRNA genes in

differ-ent plant species, we computed the neighbour distances

between every two same-strand consecutive miRNA genes

in the same chromosome The average distance of the

neighbour miRNA pairs was calculated across all

chromo-somes in the four plant species studied To evaluate the

sta-tistical significance of miRNA clustering patterns in the

four plant species, we performed a sampling approach to

evaluate significance First, we selected random positions

whose number was equal to the number of miRNA genes

on each chromosome Then we computed the neighbour

distances between consecutive random points and the

aver-age By random shuffling 1000 times, we set the P value as

the fraction of times for which the random averages were

smaller (or larger) than the average distances of miRNA

pairs to evaluate the statistical significance for clustering

patterns in the four plant species

Prediction of the core promoter of the plant

miRNA gene

The core promoter of the class II promoter, including the

TSS and the TATA-box, in the upstream sequences of plant

miRNA genes were detected using the TSSP-TCM program

(http://mendel.cs.rhul.ac.uk/mendel.php?topic=fgen) with

its default parameters; this program is well established

and is the most commonly used plant promoter prediction

software [48]

Motif analysis

To identify characteristic motifs in the flanking regions for

microRNA genes in the four plant species, we first used

RepeatMasker (version 3.2.9; http://www.repeatmasker.org)

with default settings to mask repeats in all upstream and

downstream sequences Then we applied the MEME Suite

software (version 4.3.0; http://meme.sdsc.edu/), which is a

probabilistic local alignment tool [49] The significance of

a detected motif was represented by the E-value, which

refers to the expected number of motifs of equal width

with the same or higher likelihood in a random sequence

set with the same size and nucleotide composition as the

considered set of sequences Here, MEME was used to

identify 10 top-ranking motifs for each species with a

width of 10 bp All other options were left as default Furthermore, we also applied MotifSampler, which is based on Gibbs sampling [50], to find over-represented motifs MotifSampler is a stochastic algorithm and the results may vary for different runs Therefore, we carried out 50 repeated runs of MotifSampler for each analysis The number of different motifs was set to 10 and the width of the motifs was set to 10 All other options were set at a variety of arguably sensible settings The results of these two programs were integrated to identify motifs that are frequently reported to have a low E-value among these settings and among both motif-finding tools in the flanking regions of the microRNA genes from the four plant spe-cies Sequence logos for all motifs found by these two pro-grams were created using WebLogo Version 2.8.2 (http:// weblogo.berkeley.edu) [51]

In order to determine whether a motif is statistically sig-nificant in the flanking regions of plant miRNA genes, whole-genome Monte Carlo simulation, resulting in a Z-score, was used to take into account the specificity and significance of a motif, as previously described by Zhou et al [11] For a given motif, we first obtained the average number of occurrences per target sequence, denoted

as Nt, and then randomly generated the same number of ref-erence sets from protein-coding genes and an intergenic sequence, far upstream of the miRNA, as an appropriate background Next, the MEME motifs were individually aligned using the MAST program with default values [52] to the reference sets to compute the average number of occur-rences of a motif, Nr, and its standard deviation, rr, over the reference sets The Z-score was computed as Z = (Nt⁄ Nr) = rr, which measures the normalized difference between the average occurrence of the motif in the target set and the sample mean in the reference sets [11]

Acknowledgements This work was supported in part by the National Natural Science Foundation of China (grant nos

30871394, 30600367 and 30571034), the National High Tech Development Project of China, the 863 Program (grant nos 2007AA02Z329), the National Basic Research Program of China, the 973 Program (grant nos 2008CB517302) and the National Science Foundation of Heilongjiang Province (grant nos ZJG0501, 1055HG009, GB03C602-4, JC2007H and BMFH060044)

References

1 Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs Cell 136, 669–687

2 Chen X (2008) MicroRNA metabolism in plants Curr Top Microbiol Immunol 320, 117–136