Mol. Cells 2017; 40(8): 587-597
Published online August 10, 2017
https://doi.org/10.14348/molcells.2017.0086
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: cshin@snu.ac.kr
MicroRNAs (miRNAs) are essential small RNA molecules that regulate the expression of target mRNAs in plants and animals. Here, we aimed to identify miRNAs and their putative targets in
Keywords development, flowering initiation,
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by posttranscriptional silencing mechanisms in plants and animals and control development, disease resistance, and stress responses in plants (Casadevall et al., 2013; Li et al., 2010; Wu et al., 2006; Zhu and Helliwell, 2011). Plant miRNA genes are transcribed by RNA polymerase II as primary miRNAs that are processed to precursor miRNAs (pre-miRNAs) via Dicer-like 1 (DCL1) in the nucleus. Pre-miRNAs are exported to the cytoplasm where DCL1 cleaves the pre-miRNAs near its stem-loop, giving rise to the characteristic 21–23 nucleotide (nt) long double-strand RNAs with two nucleotides 3′ overhangs. These miRNA duplexes are incorporated into the Argonaute (AGO) protein to form the effector complex, which is referred to as the RNA-induced silencing complex (RISC). The two strands of the miRNA duplex are then separated in AGO, with one strand removed and the other one retained in RISC for target recognition and silencing. In plants, most miRNAs exhibit near-perfect complementarity to their targets, often resulting in mRNA cleavage (Rogers and Chen, 2013). Because of the small size, low abundance, and tissue- or developmental stage-specific expression patterns of miRNAs, coupled with stress-induced dynamic changes in their expression, experimentally validating miRNAs has been challenging. Since the advent of next-generation sequencing, high-throughput sequencing data have become available, and computational approaches of processing large-scale data have led to the discovery of a large number of miRNA sequences (Hwang et al., 2013; Kim et al., 2012).
Taking advantage of the recent genome sequencing in
Total RNAs were extracted from
The pipeline of miRNA identification was written in Python language, and the details of the pipeline are shown in
For miRNA target validation, gene-specific 5′ RLM-RACE was performed as previously described (Hwang et al., 2013). In brief, 5 μg of pooled total RNA obtained from leaf, root, flower, and ovary tissues were ligated to 5′ the GeneRacer RNA adapter (Invitrogen). Reverse transcription producing cDNAs were synthesized with both Oligo (dT) primers and random hexamers. Primary PCR was performed with the GeneRacer 5′ primer and 3′ reverse primers containing each respective gene-specific sequence (
Small RNA libraries from leaf, root, flower and ovary were constructed and processed using a computation pipeline, as described in
Aligned small RNA sequences were analyzed to identify candidate miRNAs on the basis of the strict criteria for annotation of plant miRNA (Meyers et al., 2008). To classify conserved and novel miRNAs in
There are many miRNA copies in the
As normalized to RP10M, expression patterns of conserved and novel miRNAs from four tissues are shown in Fig. 3. First, Figs. 3A and 3B both represent conserved miRNAs with at least two-fold differences between their respective minimum and maximum expressions. Second, the top 10 most highly expressed miRNAs are shown in Fig. 3A, and the others are shown in Fig. 3B. Third, novel miRNAs are shown in Fig. 3C. Finally, some miRNAs that are consistently expressed throughout all four tissues are shown in Fig. 3D. Overall, many conserved miRNAs are expressed higher than novel miRNAs with miR156a-f-3p, miR319a-j-3p, miR390a-o-5p, miR393a-n-5p, and miR396a-i-5p being the most highly expressed (Table 2). The only highly expressed novel miRNA was miR-n001a-b-3p with 92531 reads in leaf tissues. These results indicate that conserved miRNAs are expected to play more significant roles than novel miRNAs in regulatory pathways and have a much wider range of dynamic expression for various regulations.
Many conserved miRNAs show tissue-specific expression patterns. For example, miR156a-g-5p expression was distinctively higher in the leaf and root than in the flower and ovary (Fig. 3A). Another member of miR156, miR156h-p-5p, was not highly expressed in the leaf, but it was highly expressed in the root (Fig. 3A). The differential expression of these may indicate that their biological roles are slightly different, even within the same family members. In contrast, miR172a-h-3p was exclusively expressed in the flower and ovary. Previous studies showed that miR156 is an upstream regulator of miR172 and acts by repressing miR172 through its target,
miR396a-i-5p expression in the leaf was much higher than that in other tissues (Fig. 3A). miR396 regulates the
Similar to miR172, miR319a-j-3p expression was much higher in the flower and ovary than in the leaf and root (Fig. 3A), whereas miR319k-n-3p expression (Fig. 3B) was higher in the root than in any other tissues. Previous studies showed that the loss-of-function miR319 mutants of
Some miRNAs were consistently expressed in all four tissues (Fig. 3D). For instance, miR166a-ag-3p showed quite high expression patterns in every tissue. Mutation studies demonstrated that miR166 overexpression affected several class III homeodomain-leucine zipper family genes, possibly causing defects in the shoot meristem, leaves, gynoecia, and vascular tissues of
Novel miRNAs generally showed lower and less dynamic expression than those of conserved miRNAs (Figs. 3A–3C), suggesting their species-specific roles in particular circumstances. However, a few novel miRNAs exhibited dynamic expression patterns; for example, miR-n001a-b-3p and miR-n006 family genes were highly expressed in leaf, whereas miR-n003a-e-3p displayed the highest expression in flower. These results may indicate tissue-specific roles of some novel miRNA families.
To investigate the potential regulatory roles of miRNAs in
Noticeable targets of conserved miRNAs include many transcription factors such as
In this study, we first discovered a novel target of the conserved miRNA, miR477. miR477c-e-5p was predicted to regulate the terpene synthase gene, which was validated by 5′ RLM-RACE analysis (Fig. 4E). Terpene synthase is an enzyme for synthesizing terpene, which is associated with the plant innate immunity (Wang et al., 2012). Thus, we expect these results to provide a foundation for further studies regarding the control of terpene synthesis. The recent genome study of
As described above, we proposed that WGD might affect the expansion of some miRNA families and protein-coding genes in
Novel miRNA targets include pentatricopeptide repeat-containing (PPR) proteins, serine/threonine-protein kinase, receptor-like protein (RLP) and transcription factor MYC2-like. Most novel miRNAs exhibited weaker expressions than conserved miRNAs. The only exception was miR-n001a-b-3p, which is expressed as much as conserved miRNAs (Table 3), indicating that miR-n001a-b-3p may have an important biological role in
In contrast to conserved miRNAs, we were unable to validate some targets of novel miRNAs via the 5′ RLM-RACE analysis (data not shown). It is in agreement with earlier studies that the targets of novel miRNAs are generally not identifiable (Hwang et al., 2013; Moxon et al., 2008). There are possible reasons as follows: (1) the abundance of most novel miRNAs were not high enough to direct the cleavage of mRNA targets, (2) target mRNAs and miRNAs were not colocalized or coexpressed (Cuperus et al., 2011), (3) the mode of novel miRNA action is repression at the protein level (Aukerman and Sakai, 2003), (4) the gene annotation of
In this study, we discovered 93 conserved and 36 novel miRNA sequences from a small RNA library of the novel species
Mol. Cells 2017; 40(8): 587-597
Published online August 31, 2017 https://doi.org/10.14348/molcells.2017.0086
Copyright © The Korean Society for Molecular and Cellular Biology.
Taewook Kim1, June Hyun Park1, Sang-gil Lee2, Soyoung Kim1, Jihyun Kim2, Jungho Lee3, and Chanseok Shin1,4,5,*
1Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea, 2Program in Applied Life Chemistry, Seoul National University, Seoul,08826, Korea, 3Green Plant Institute, Yongin 16954, Korea, 4Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea, 5Plant Genomics and Breeding Institute, Seoul National University, Seoul 08826, Korea
Correspondence to:*Correspondence: cshin@snu.ac.kr
MicroRNAs (miRNAs) are essential small RNA molecules that regulate the expression of target mRNAs in plants and animals. Here, we aimed to identify miRNAs and their putative targets in
Keywords: development, flowering initiation,
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by posttranscriptional silencing mechanisms in plants and animals and control development, disease resistance, and stress responses in plants (Casadevall et al., 2013; Li et al., 2010; Wu et al., 2006; Zhu and Helliwell, 2011). Plant miRNA genes are transcribed by RNA polymerase II as primary miRNAs that are processed to precursor miRNAs (pre-miRNAs) via Dicer-like 1 (DCL1) in the nucleus. Pre-miRNAs are exported to the cytoplasm where DCL1 cleaves the pre-miRNAs near its stem-loop, giving rise to the characteristic 21–23 nucleotide (nt) long double-strand RNAs with two nucleotides 3′ overhangs. These miRNA duplexes are incorporated into the Argonaute (AGO) protein to form the effector complex, which is referred to as the RNA-induced silencing complex (RISC). The two strands of the miRNA duplex are then separated in AGO, with one strand removed and the other one retained in RISC for target recognition and silencing. In plants, most miRNAs exhibit near-perfect complementarity to their targets, often resulting in mRNA cleavage (Rogers and Chen, 2013). Because of the small size, low abundance, and tissue- or developmental stage-specific expression patterns of miRNAs, coupled with stress-induced dynamic changes in their expression, experimentally validating miRNAs has been challenging. Since the advent of next-generation sequencing, high-throughput sequencing data have become available, and computational approaches of processing large-scale data have led to the discovery of a large number of miRNA sequences (Hwang et al., 2013; Kim et al., 2012).
Taking advantage of the recent genome sequencing in
Total RNAs were extracted from
The pipeline of miRNA identification was written in Python language, and the details of the pipeline are shown in
For miRNA target validation, gene-specific 5′ RLM-RACE was performed as previously described (Hwang et al., 2013). In brief, 5 μg of pooled total RNA obtained from leaf, root, flower, and ovary tissues were ligated to 5′ the GeneRacer RNA adapter (Invitrogen). Reverse transcription producing cDNAs were synthesized with both Oligo (dT) primers and random hexamers. Primary PCR was performed with the GeneRacer 5′ primer and 3′ reverse primers containing each respective gene-specific sequence (
Small RNA libraries from leaf, root, flower and ovary were constructed and processed using a computation pipeline, as described in
Aligned small RNA sequences were analyzed to identify candidate miRNAs on the basis of the strict criteria for annotation of plant miRNA (Meyers et al., 2008). To classify conserved and novel miRNAs in
There are many miRNA copies in the
As normalized to RP10M, expression patterns of conserved and novel miRNAs from four tissues are shown in Fig. 3. First, Figs. 3A and 3B both represent conserved miRNAs with at least two-fold differences between their respective minimum and maximum expressions. Second, the top 10 most highly expressed miRNAs are shown in Fig. 3A, and the others are shown in Fig. 3B. Third, novel miRNAs are shown in Fig. 3C. Finally, some miRNAs that are consistently expressed throughout all four tissues are shown in Fig. 3D. Overall, many conserved miRNAs are expressed higher than novel miRNAs with miR156a-f-3p, miR319a-j-3p, miR390a-o-5p, miR393a-n-5p, and miR396a-i-5p being the most highly expressed (Table 2). The only highly expressed novel miRNA was miR-n001a-b-3p with 92531 reads in leaf tissues. These results indicate that conserved miRNAs are expected to play more significant roles than novel miRNAs in regulatory pathways and have a much wider range of dynamic expression for various regulations.
Many conserved miRNAs show tissue-specific expression patterns. For example, miR156a-g-5p expression was distinctively higher in the leaf and root than in the flower and ovary (Fig. 3A). Another member of miR156, miR156h-p-5p, was not highly expressed in the leaf, but it was highly expressed in the root (Fig. 3A). The differential expression of these may indicate that their biological roles are slightly different, even within the same family members. In contrast, miR172a-h-3p was exclusively expressed in the flower and ovary. Previous studies showed that miR156 is an upstream regulator of miR172 and acts by repressing miR172 through its target,
miR396a-i-5p expression in the leaf was much higher than that in other tissues (Fig. 3A). miR396 regulates the
Similar to miR172, miR319a-j-3p expression was much higher in the flower and ovary than in the leaf and root (Fig. 3A), whereas miR319k-n-3p expression (Fig. 3B) was higher in the root than in any other tissues. Previous studies showed that the loss-of-function miR319 mutants of
Some miRNAs were consistently expressed in all four tissues (Fig. 3D). For instance, miR166a-ag-3p showed quite high expression patterns in every tissue. Mutation studies demonstrated that miR166 overexpression affected several class III homeodomain-leucine zipper family genes, possibly causing defects in the shoot meristem, leaves, gynoecia, and vascular tissues of
Novel miRNAs generally showed lower and less dynamic expression than those of conserved miRNAs (Figs. 3A–3C), suggesting their species-specific roles in particular circumstances. However, a few novel miRNAs exhibited dynamic expression patterns; for example, miR-n001a-b-3p and miR-n006 family genes were highly expressed in leaf, whereas miR-n003a-e-3p displayed the highest expression in flower. These results may indicate tissue-specific roles of some novel miRNA families.
To investigate the potential regulatory roles of miRNAs in
Noticeable targets of conserved miRNAs include many transcription factors such as
In this study, we first discovered a novel target of the conserved miRNA, miR477. miR477c-e-5p was predicted to regulate the terpene synthase gene, which was validated by 5′ RLM-RACE analysis (Fig. 4E). Terpene synthase is an enzyme for synthesizing terpene, which is associated with the plant innate immunity (Wang et al., 2012). Thus, we expect these results to provide a foundation for further studies regarding the control of terpene synthesis. The recent genome study of
As described above, we proposed that WGD might affect the expansion of some miRNA families and protein-coding genes in
Novel miRNA targets include pentatricopeptide repeat-containing (PPR) proteins, serine/threonine-protein kinase, receptor-like protein (RLP) and transcription factor MYC2-like. Most novel miRNAs exhibited weaker expressions than conserved miRNAs. The only exception was miR-n001a-b-3p, which is expressed as much as conserved miRNAs (Table 3), indicating that miR-n001a-b-3p may have an important biological role in
In contrast to conserved miRNAs, we were unable to validate some targets of novel miRNAs via the 5′ RLM-RACE analysis (data not shown). It is in agreement with earlier studies that the targets of novel miRNAs are generally not identifiable (Hwang et al., 2013; Moxon et al., 2008). There are possible reasons as follows: (1) the abundance of most novel miRNAs were not high enough to direct the cleavage of mRNA targets, (2) target mRNAs and miRNAs were not colocalized or coexpressed (Cuperus et al., 2011), (3) the mode of novel miRNA action is repression at the protein level (Aukerman and Sakai, 2003), (4) the gene annotation of
In this study, we discovered 93 conserved and 36 novel miRNA sequences from a small RNA library of the novel species
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