Introns targeted by plant microRNAs: a possible novel mechanism of gene regulation
© Meng et al.; licensee Springer. 2013
Received: 22 November 2012
Accepted: 25 March 2013
Published: 15 April 2013
In plant cells, most microRNAs (miRNAs) perform cleavages of target mature mRNAs in the cytoplasm. A recent report of a miRNA pathway involved in DNA methylation in the rice nucleus raises the possibility that plant miRNAs could cleave intron-containing pre-mRNAs (the precursor of messenger RNAs) located in the nucleus.
In this study, we searched for the miRNA binding sites present within the introns of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genes. All miRNA—intron interactions predicted to result in cleavages were validated by using the public degradome sequencing data. As a result, 40 miRNA—intron pairs involving 25 miRNAs in Arabidopsis and 1912 pairs involving 91 miRNAs in rice were identified. For several rice genes, not all transcription forms (alternative splicing variants) were under similar regulation by specific miRNAs. Certain transcripts could escape cleavages due to the absence of intronic miRNA binding sites within these sequences. In some instances, specific cleaved intron remnants could be converted to double-stranded RNAs (dsRNAs) by RNA-dependent RNA polymerase 2. These dsRNAs could then be processed into 21- and 24-nt phased sRNAs by the activity of Dicer-like 1 and 3, respectively. The resultant siRNAs have the potential to be incorporated into Argonaute (AGO)-associated silencing complexes and result in cleavages of target pre-mRNA sequences.
A regulatory model, miRNA—targeting of intron-containing pre-mRNAs—phased sRNAs—targeting of mature mRNAs is proposed, which further expands the potential modes of action of plant miRNAs.
KeywordsPlant microRNA Pre-mRNA (the precursor of messenger RNA) Intron RDR (RNA-dependent RNA polymerase) DCL (Dicer-like) Phased small RNA Degradome
MicroRNAs (miRNAs) play an essential role in modulating gene expression levels in organisms (Carthew & Sontheimer 2009; Chen 2009; Jones-Rhoades et al. 2006; Voinnet 2009). In plants, miRNAs are involved in many essential biological processes, such as morphogenesis, hormone signaling, and developmental phase transition (Chen 2009; Jones-Rhoades et al. 2006). Mediated by RNA polymerase II, a miRNA gene (Lee et al. 2004; Xie et al. 2005) is firstly transcribed into pri-miRNA (primary microRNA). After two-step cleavages by Dicer-like 1 (DCL1), the pri-miRNA is processed into the pre-miRNA (precursor microRNA), then into a miRNA/miRNA* duplex. The duplex is exported to the cytoplasm, and the miRNA strand is selectively incorporated into specific Argonaute (AGO)-associated gene silencing complex. Based on the well-established model, a dominant portion of the plant miRNAs are associated with AGO1. These miRNAs could guide the silencing complexes to bind the transcripts containing highly complementary recognition sites. Then, site-specific cleavages could be performed in the cytoplasm (Jones-Rhoades et al. 2006; Voinnet 2009). In this regard, the mature mRNAs (messenger RNAs), but not the pre-mRNAs (the precursor of messenger RNA), of the protein-coding genes, should be the miRNA targets. However, a recent report in rice showed us that some of the 24-nt miRNAs associated with AGO4 complexes could mediate DNA methylation in the nucleus (Wu et al. 2010). This raises the possibility of cleavages of the nuclear-localized, intron-containing pre-mRNAs by certain AGO1-associated miRNAs.
In this study, we performed a large-scale search for the miRNA binding sites present within the introns of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). All the cleavage-based miRNA—intron interactions were validated by using public degradome sequencing data. As a result, 40 miRNA—intron pairs in Arabidopsis, and 1912 miRNA—intron pairs in rice were identified. Several interesting phenomena were observed. In some cases in rice, certain transcription forms (alternative splicing variants) of a gene could escape from the regulation by specific miRNA due to the absence of the binding sites within the intron. Also in rice, some cleaved intron remnants could be converted to double-stranded RNAs (dsRNAs) by RNA-dependent RNA polymerase 2 (RDR2). These dsRNAs could be further processed into 21- and 24-nt phased small RNAs (sRNAs) relying on the activity of DCL1 and DCL3, respectively. Some of the phased sRNAs could be incorporated into AGO1 or AGO4 clade proteins. Some of the AGO1-associated sRNAs possessed great potential to perform site-specific target cleavages. Based on the above results, a novel mode of action of the plant miRNAs in the nucleus, involving the regulatory cascade miRNA—intron—secondary phased sRNAs—targets, was proposed. We hope that this study inspires further research efforts on thorough elucidation of this novel action mode, which could advance the current understanding on the miRNA activities in plants.
Results and discussion
Identification of miRNA—intron pairs
The annotated introns of Arabidopsis and rice were collected from the Arabidopsis information resource (TAIR, release 10) (Huala et al. 2001) and the rice genome annotation project established by the institute for genome research (TIGR rice, release 6.1) (Yuan et al. 2003), respectively. All the introns with 30-nt flanking sequences at both ends were subjected to miRNA (miRBase (Griffiths-Jones et al. 2008), release 18) binding site prediction by using miRU algorithm (Dai & Zhao 2011; Zhang 2005). Plant degradome sequencing is quite efficient for the identification of miRNA cleavage sites on the targets (Addo-Quaye et al. 2008; German et al. 2009; German et al. 2008; Li et al. 2010; Pantaleo et al. 2010). In this regard, the predicted miRNA—intron pairs were subjected to degradome sequencing data-based validation by employing target plots as previously described (German et al. 2009; German et al. 2008; Meng et al. 2011). The publicly available degradome sequencing data sets (11 in Arabidopsis and 4 in rice) were utilized (see details in Methods). As a result, 40 miRNA—intron interactions involving 25 miRNAs in Arabidopsis and 1912 interactions involving 91 miRNAs in rice were obtained (Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3 and Additional file 4: Figure S4, and Additional file 5: Table S1 and Additional file 6: Table S2). The number of the miRNA—intron pairs identified in Arabidopsis is much less than that in rice. This discrepancy is likely resulted from the smaller numbers of the annotated introns and the registered miRNAs in Arabidopsis when compared to those in rice (175,512 introns in Arabidopsis vs. 251,812 in rice; 328 miRNAs in Arabidopsis vs. 661 in rice). Together, these results indicate that certain plant miRNAs might perform cleavages on the intron-containing pre-mRNAs in the nucleus, which needs further validation.
Certain transcription form(s) of a rice gene could escape from the regulation of a specific miRNA due to the lack of the miRNA binding site
RDR2-dependent synthesis of dsRNAs from the cleaved intron remnants and DCL1/3-dependent production of phased sRNAs
The introns are spliced out and subject to degradation during mRNA maturation. However, based on our previous study, huge numbers of short sequences generated by sRNA high-throughput sequencing (HTS) could be mapped to the introns (data not shown). Focusing on our study, one question was raised here: once sliced by specific miRNAs, were the cleaved intron remnants totally subjected to degradation? Referring to the biogenesis model of the ta-siRNA (-acting small interfering RNA) (Allen et al. trans2005; Williams et al. 2005), we hypothesized that the cleaved remnants might be converted to dsRNAs, which could be further processed into phased secondary sRNAs through DCL-mediated dicing.
To test this possibility, firstly, the short sequences from the publicly available sRNA HTS data sets (see details of these HTS data sets in Additional file 8: Table S3 and Additional file 9: Table S4) were mapped onto the miRNA-regulated introns (see intron IDs in Additional file 5: Table S1 and Additional file 6: Table S2). Only the perfectly matched sRNAs were retained. Then, a Perl script was developed to search for the phase-distributed sRNAs within the introns. The search criteria were set as follows: (1) A defined set of phased sRNAs, it should be constituted by three or more continuous sRNA duplexes with constant sequence length (defined as phase step), such as 21 nt. (2) According to the enzymatic feature of DCLs, each sRNA duplex diced from the dsRNA synthesized from an intron should possess 2-nt 3’ overhangs at both ends. (3) Considering the possibility that the HTS data utilized in this study might not reflect the expression levels of all the phased sRNAs, the phased sRNA duplex with only one detectable strand was allowed. (4) For the overlapping sRNA sets sharing the same phase step, the longest set was retained for the analysis.
As a result, thousands of phased sRNA sets were discovered within the introns of rice, but not of Arabidopsis. According to the biogenesis model of ta-siRNAs, the miRNA cleavage sites on the target transcripts could define the start points of the phased sRNA sets (Allen et al. 2005; Williams et al. 2005). In this regard, another Perl script was developed to search for the phased sRNA sets with either ends resided within the intronic miRNA binding sites validated based on the degradome sequencing data (Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3 and Additional file 4: Figure S4). Interestingly, for most of the sRNA sets identified, the corresponding miRNA binding sites located at the 5’ ends of these sets (Additional file 10: Data S1). This observation fits well with the notion that the 3’ remnants cleaved by specific miRNAs are more stable than the 5’ remnants.
The above results only indicate the possibility that the introns cleaved by specific miRNAs might be converted to dsRNAs for the production of phased sRNAs. We attempted to obtain more evidences. Considering the fact that the synthesis of the dsRNAs depends on RDR(s), and the production of the phased sRNAs relies on DCL(s), we utilized the following sRNA HTS data sets for further analysis: GSM520640 (prepared from the wild type rice seedlings, served as the control set for GSM520639, GSM520637 and GSM520638), GSM520639 (prepared from the rice mutant rdr2), GSM520637 (prepared from the rice mutant dcl1), GSM520638 (prepared from the rice mutant dcl3), GSM455965 (total RNA extracts from rice seedlings, served as the control set for the following six data sets), GSM455962 (sRNAs associated with the rice AGO1a protein), GSM455963 (sRNAs associated with the rice AGO1b protein), GSM455964 (sRNAs associated with the rice AGO1c protein), GSM520634 (sRNAs associated with the rice AGO4a protein), GSM520635 (sRNAs associated with the rice AGO4b protein) and GSM520636 (sRNAs associated with the rice AGO16 protein). The accumulation levels of each sRNA belonging to a phased set were calculated for all the above sRNA HTS data sets.
miRNA—intron—phased sRNAs—targets: a possible novel regulatory cascade reflecting a new mode of action of the plant miRNAs
According to the canonical model of miRNA biogenesis and action in plants (Jones-Rhoades et al. 2006; Voinnet 2009), the miRNA transcripts were processed and exported to the cytoplasm. The miRNA strands were then incorporated into the AGO1-associated silencing complexes, guiding the complexes to bind targets such as the mature mRNAs. Then, the target transcripts were cleaved by the AGO1 complexes in the cytoplasm. However, a recent study by Qi’s group demonstrated that certain miRNAs could mediate DNA methylation in the plant nucleus (Wu et al. 2010). In this regard, we hypothesized that some of the AGO1-associated miRNAs regulate the intron-containing pre-mRNAs in the nucleus. In this study, the possibility of the nuclear-localized interactions between the miRNAs and the introns was tested in two model plants. Especially in rice, dozens of predicted miRNA binding sites on the introns were supported by the degradome sequencing data.
Thus, we propose a novel additional regulatory cascade “miRNA—intron—phased secondary sRNAs—targets”. However, much more experimental efforts are needed to thoroughly elucidate this possible novel pathway. For example, we should experimentally exclude the possibility that the observed intronic cleavage signals originated from the retained (unspliced) introns in the cytoplasm. We hope that our study would advance the current understanding on the modes of action of miRNAs in plants.
The annotated transcription forms, the introns, and the gene annotations of Arabidopsis were retrieved from the FTP site of the Arabidopsis information resource (TAIR, release 10; ftp://ftp.arabidopsis.org/home/tair/Sequences/blast_datasets/TAIR10_blastsets/) (Huala et al. 2001), and those of rice were obtained from the FTP site of the rice genome annotation project established by the institute for genome research (TIGR rice, release 6.1; ftp://ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_6.1/) (Yuan et al. 2003).
For the Arabidopsis degradome sequencing data sets, GSM278333, GSM278334, GSM278335 and GSM278370 were retrieved from Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) (Barrett et al. 2009), and AxIDT, AxIRP, AxSRP, Col, ein5l, TWF and Tx4F were retrieved from Arabidopsis PARE Database (http://mpss.udel.edu/at_pare/) (Nakano et al. 2006). For rice, the degradome data sets GSM434596, GSM455938, GSM455939 and GSM476257 were retrieved from GEO.
The sRNA HTS data sets of Arabidopsis and rice were downloaded from GEO, Next-Gen Sequence Databases (http://mpss.udel.edu/) (Nakano et al. 2006), and Cereal Small RNAs Database (CSRDB; http://sundarlab.ucdavis.edu/smrnas/) (Johnson et al. 2007). See Additional file 8: Table S3 and Additional file 9: Table S4 for detailed information.
Prediction and validation of the intron targets of the miRNAs
Target prediction was performed by using miRU algorithm (Dai & Zhao 2011; Zhang 2005) with default parameters. The degradome sequencing data were utilized to validate the predicted miRNA—intron pairs. First, in order to allow cross-library comparison, the normalized read count (in RPM, reads per million) of a short sequence belonging to a specific degradome library was calculated by dividing the raw count of this sequence by the total counts of the library, and then multiplied by 106. Then, two-step filtering was performed to extract the most likely miRNA—intron interactions. During the first step, the predicted miRNA binding sites along with the 50-nt surrounding sequences at both ends were collected. For the BLAST, all the collected degradome data sets were utilized at the same time to do a comprehensive search. It was based on the scenario that a miRNA—intron pair was considered to be the candidate once the cleavage signals were detected in any data set(s). The introns meeting the following criteria were retained: (1) there must be perfectly matched degradome signatures with their 5’ ends resided within 8—14 nt region away from the 5’ ends of the miRNAs; and (2) for a specific position within the 8—14 nt region (regarded as the potential cleavage site) there must be two or more distinct degradome signatures to support this position. These retained transcripts were subjected to a second BLAST, and the degradome signals along each full-length gene were obtained to provide a global view of the signal noise when compared to the signal intensity within a specific binding site. Referring to our previous study (Meng et al. 2011), the target plots (German et al. 2009; German et al. 2008) were drawn for the subsequent manual filtering. Only the introns with cleavage signals easy to be recognized were extracted as the miRNA—intron pairs.
The precursor of messenger RNA
RNA-dependent RNA polymerase
The Arabidopsis information resource
The institute for genome research
Squamosa-promoter binding protein-like
YELLOW STRIPE like 3
Trans-acting small interfering RNA
Gene Expression Omnibus
Cereal Small RNAs Database
Reads per million
We would like to thank all the publicly available data sets and the scientists behind them. We would like to thank the anonymous reviewers for their constructive comments.
This work was funded by the National Natural Sciences Foundation of China  and , the Starting Grant funded by Hangzhou Normal University to Yijun Meng [2011QDL60].
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