Genome-wide analysis of clustering patterns and flanking characteristics for plant microRNA genes Meng Zhou1,*, Jie Sun1,*, Qiang-Hu Wang1,*, Li-Qun Song2, Guang Zhao1, Hong-Zhi Wang2, Hai-Xiu Yang1 and Xia Li1 College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China Department of Internal Medicine, Affiliated Hospital of Heilongjiang University of Chinese Medicine, Harbin, China 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 December 2010, accepted 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 < 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 algorithms to search for over-represented, statistically significant sequence motifs, and discovered six species-specific motifs across the four plant species studied Moreover, we also identified, for the first time, several significantly 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 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 discovered 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 (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 orthologue HASTY and other unknown factors Mature RNA duplexes excised from pre-miRNAs (miRNA ⁄ miRNA*, where miRNA is the guide strand and miRNA* is the degraded strand) are methylated by HEN1 The guide miRNA strand is then incorporated Abbreviations miRNA, microRNA; Pol II, RNA polymerase II; pri-miRNAs, primary miRNA transcripts; TSSs, transcription start sites FEBS Journal 278 (2011) 929–940 ª 2011 The Authors Journal compilation ª 2011 FEBS 929 Clustering and flanking characteristics for plant miRNAs M Zhou et al 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 intragenic miRNAs are possibly transcribed with, or independently of, the host genes Recently, Heikkinen et al [13] examined the upstream sequences of miRNAs in C elegans and Caenorhabditis briggsae, and discovered a sequence motif – GANNNNGA – common 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 characteristics of the upstream sequences of miRNA genes in animals in an attempt to understand transcriptional regulation [11,13–16], similar analyses have been performed 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 genetics, direct cloning and computational prediction An increasing number of plant miRNAs provide a good opportunity to uncover complex transcriptional regulation mechanisms for plant miRNAs In our study, we performed computational approaches, based on genome-wide analyses, to examine the clustering patterns of plant miRNAs In addition, we analyzed regions, up to kb upstream and up to kb downstream, 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 processing of plant miRNAs and provide useful knowledge for understanding the mechanism of the origin and computational identification of new miRNAs in plants 930 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 miRBase coordinates The cumulative distance distribution of the miRNA gene pairs is presented in Fig and shows that 17.71%, 26.94% and 29.07% of the miRNA 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 Furthermore, 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 proteincoding gene pairs were separated by very short distances of < kb To evaluate the statistical significance 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 distances, as described in the Materials and methods, and found that the miRNA gene pair distances were statistically significantly higher than expected at random (P < 0.001) To identify more characteristics of miRNA 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 According 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 clusters at very short gene-pair distances in O sativa and Sorghum bicolor was significantly larger than in A thaliana 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 specific clustering pattern of miRNAs may be indicative of functional divergence of the miRNA cluster in FEBS Journal 278 (2011) 929–940 ª 2011 The Authors Journal compilation ª 2011 FEBS M Zhou et al Clustering and flanking characteristics for plant miRNAs Fig 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 logarithmic scale 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 kb in the zebrafish genome [22] In contrast to patterns of clustering found in animal miRNAs, only a small proportion of plant miRNAs (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 polycistronic 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 polycistronic 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 identified 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 FEBS Journal 278 (2011) 929–940 ª 2011 The Authors Journal compilation ª 2011 FEBS 931 932 2.69 3.11 1.82 1.86 2.57 2.35 3.12 2.67 54 40 103 32 21 17 33 12 19.72% 14.1% 19.48% 18.24% 1.40 2.33 1.08 1.14 2.47 2.36 3.21 2.7 42 33 90 27 17 14 28 10 15.02% 7.69% 16.02% 12.16% 0.67 0.45 0.48 0.36 2.13 2.85 32 18 74 18 15 26 The number of miRNA genes studied in four plant species b The number of predicted clusters c The number of miRNA genes located in clusters d The average number of miRNA genes in a cluster for the four plant species e The average distance between two miRNA genes in a cluster f The percentage of miRNA genes located in clusters a 213 234 462 148 A thaliana P trichocarpa O sativa S bicolor 25.35% 17.09% 22.29% 21.62% miRNAsa Clusterb Membersc Averaged Distancese Percentagef Cluster Members Average Distances Percentage Cluster Members Average Distances Percentage Species kb Table Characterization of miRNA clusters in four plant species kb 10 kb Clustering and flanking characteristics for plant miRNAs M Zhou et al 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 architecture of different miRNAs in plants and the relationship between the number of Pol II promoters and the degree of conservation of miRNAs, we classified four plant miRNA genes into two types (intergenic miRNAs and intragenic miRNAs) based on their genomic locations Then, the miRNAs from the four plant species studied were divided into three groups (based on evolutionary conservation across all plant species, as described in the Materials and methods): highly conserved miRNAs, low conserved miRNAs and nonconserved miRNAs The results are summarized in Fig 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 indicate 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 independent unit from their own promoter However, in plants, a small number of miRNAs with no class II promoter may be transcribed through other transcriptional 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 significantly higher than in low conserved (P < 0.001; twosample t-test) and in nonconserved (P < 0.001; twosample 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 conserved 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 conFEBS Journal 278 (2011) 929–940 ª 2011 The Authors Journal compilation ª 2011 FEBS M Zhou et al Clustering and flanking characteristics for plant miRNAs S bicolor A Intragenic 50% O sativa 50% Intragenic Intergenic Intergenic 40% 40% 30% 30% 20% 20% 10% 10% 0% ≥3 The number of core promoter 0% P trichocarpa A thaliana 60% ≥3 The number of core promoter Intragenic 50% Intragenic Intergenic 50% Intergenic 40% 40% 30% 30% 20% 20% 10% 10% 0% ≥3 The number of core promoter 0% ≥3 The number of core promoter B 100% 80% Non-conserved 60% Low conserved Fig Distribution of miRNA genes with the same number of putative core promoters (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 Highly conserved 40% 20% 0% ≥3 The number of core promoter served miRNAs (P < 0.001; Fisher’s exact test) and 23.94% of nonconserved miRNAs (P < 0.0001; Fisher’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 conserved 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 obvious effects on plant morphology [30] Therefore, we speculate that the increased number of Pol II promoters 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 FEBS Journal 278 (2011) 929–940 ª 2011 The Authors Journal compilation ª 2011 FEBS 933 Clustering and flanking characteristics for plant miRNAs M Zhou et al A S bicolor 50% O sativa 40% A thaliana P trichocarpa 30% 20% 10% 0% –0.4 kb –0.8 kb –1.2 kb –1.6 kb –2 kb B Highly conserved 40% Low conserved Non-conserved 30% 20% 10% 0% –0.4 kb –0.8 kb –1.2 kb –1.6 kb –2 kb Fig 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 corresponding miRNA stem–loop sequences, and the vertical axis shows the percentage of putative core promoters at the specified positions (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 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 distributed mainly from to 0.4 kb and from 1.6 to kb Together, these results indicate that this distribution pattern of putative core promoters seems to be conserved in the 2-kb region upstream of miRNAs in different 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 400bp region upstream of the miRNA However, core promoters are distributed mainly in the 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 promoters 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 conservation in plants Based on these observations, we propose 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 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 934 To further identify specific characteristic motifs in the flanking regions of miRNAs in four plant species, we performed motif analysis to search for over-represented and statistically significant motifs in the flanking regions up to kb upstream and 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 significance of motifs identified, as described in the Materials and methods Motifs whose Z-scores were > 2.0 were considered as over-represented and statistically significant motifs Several significantly over-represented species-specific motifs were identified in the flanking regions of four plant species All the species-specific FEBS Journal 278 (2011) 929–940 ª 2011 The Authors Journal compilation ª 2011 FEBS M Zhou et al Clustering and flanking characteristics for plant miRNAs motifs found in the four plant species studied are shown in Table 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 species-specific motifs, we compared our species-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 consensus 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 GCArepeats 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 promoters 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 networks 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 Significantly over-represented species-specific sequence motifs identified in the flanking regions of the three plant species studied Species Index Consensus sequencea A thaliana M1 Z-scored GGCCTGAGCC 1.4e-008 10.62 TTAGGGTTTC 2.4e-009 4.31 M3 GCAGCAGAAG 7.2e-006 6.21 M4 CGGGTCAAAC 3.6e-016 4.45 M5 GCATGCATGC 2.7e-030 5.86 M6 S bicolor E-valuec M2 P trichocarpa Motif logob GAACTAAACA 2.1e-019 3.53 a The consensus sequence represents a sequence of the most frequent base at each position b The motif logos show the information content 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 FEBS Journal 278 (2011) 929–940 ª 2011 The Authors Journal compilation ª 2011 FEBS 935 Clustering and flanking characteristics for plant miRNAs M Zhou et al The mechanism by which new plant miRNAs originate is not fully understood It is believed that the origin 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 Two motifs (CATGCATGCA and CTAGCTAGCT; M1 and M2, 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 considered as CATG repeats and CTAG repeats, respectively However, we did not find any significantly overrepresented 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 multicopy [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 significantly over-represented sequence motifs in highly conserved and low conserved 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 processing of plant miRNAs, including the miRNAs located in the intergenic area and in the protein-coding area whose possible sequence characteristics were not studied earlier Previous studies have revealed that miRNAs 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 statistically 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 Significantly over-represented sequence motifs related to the conservation of miRNAs Conservation Index Consensus sequencea Highly M1 E-valuec Z-scored CATGCATGCA 3.6e-019 7.82 M2 Low Motif logob CTAGCTAGCT 1.6e-024 5.76 M3 TGGCGGGAAA 24e-014 4.32 a The consensus sequence represents a sequence of the most frequent base at each position b The motif logos show the information content 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 936 FEBS Journal 278 (2011) 929–940 ª 2011 The Authors Journal compilation ª 2011 FEBS M Zhou et al Clustering and flanking characteristics for plant miRNAs protein-coding genes in the very short pairwise distances of < 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 nonconserved miRNAs We applied two motif-finding tools to search for over-represented, statistically significant 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 speciesspecific motifs and some novel species-specific motifs We also identified three specific motifs associated with the degree of miRNA conservation Compared with previous studies, our study systematically explored clustering patterns and the characteristics of flanking regions up to kb upstream and 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 conservation 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 understanding of transcriptional regulation and processing 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 species were downloaded from the miRBase Sequence Database, 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 kb upstream and up to 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 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 homology search for known miRNAs from the four plants to detect both closely related and distantly related homologues 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 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 A thaliana P trichocarpa O sativa S bicolor TAIR9 JGI_Poptr2.0 MSU6.0 JGI_sbi1 213 234 462 148 21 17 33 12 167 163 326 125 167 163 326 125 FEBS Journal 278 (2011) 929–940 ª 2011 The Authors Journal compilation ª 2011 FEBS 937 Clustering and flanking characteristics for plant miRNAs M Zhou et al variations [19] Finally, we divided the miRNAs of the four plant species into three groups: the miRNAs whose homologues were found simultaneously in monocots and eudicots were considered as highly conserved miRNAs; those found only in monocots or eudicots were considered as low conserved 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 different 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 chromosomes in the four plant species studied To evaluate the statistical 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 average 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 938 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 species Sequence logos for all motifs found by these two programs were created using WebLogo Version 2.8.2 (http:// weblogo.berkeley.edu) [51] In order to determine whether a motif is statistically significant 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 reference 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 occurrences 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 Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs Cell 136, 669–687 Chen X (2008) MicroRNA metabolism in plants Curr Top Microbiol Immunol 320, 117–136 FEBS Journal 278 (2011) 929–940 ª 2011 The Authors Journal compilation ª 2011 FEBS M Zhou et al Ambros V (2004) The functions of animal microRNAs Nature 431, 350–355 Singh SK, Pal Bhadra M, Girschick HJ & Bhadra U (2008) MicroRNAs–micro in size but macro in function Febs J 275, 4929–4944 Lee RC, Feinbaum RL & Ambros V (1993) The C elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 Cell 75, 843– 854 Park W, Li J, Song R, Messing J & Chen X (2002) CARPEL FACTORY, a Dicer homolog, and 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[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... 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. .. understanding of transcriptional regulation and processing of plant miRNAs and provide useful knowledge for understanding the mechanism of the origin and computational identification of new miRNAs in plants