Elsevier

Gene

Volume 527, Issue 2, 25 September 2013, Pages 477-483
Gene

Methods paper
Identification of novel microRNA-like-coding sites on the long-stem microRNA precursors in Arabidopsis

https://doi.org/10.1016/j.gene.2013.06.070Get rights and content

Highlights

  • We did a comprehensive search for miRNA candidates on the pre-miRNAs of Arabidopsis.

  • AGO1 enrichment, miRNA*-like coordinates, and unique genomic sites were considered.

  • As a result, 43 miRNA-like candidates deriving from 25 pre-miRNAs were identified.

  • Certain miRNA candidates showed organ-specific expression patterns.

  • Five miRNA–target pairs were identified, providing functional hints.

Abstract

Plant microRNA (miRNA) is a crucial regulator of gene expression. It has been reported that more than one miRNA/miRNA* duplex could be produced from a microRNA precursor (pre-miRNA). In this study, we performed a comprehensive search for the novel miRNA candidates on the pre-miRNAs of Arabidopsis. AGO1 enrichment, co-existence of the miRNA*-like coordinates, and unique genome-wide match sites were taken into consideration for candidate screening. As a result, 43 miRNA-like candidates derived from 25 pre-miRNAs were identified. Among these candidates, 31 strong candidates from 22 pre-miRNAs passed all the filtering steps. Interestingly, some of these miRNA-like candidates showed organ-specific expression patterns. After target prediction and degradome sequencing data-based validation, five miRNA candidate–target pairs (ath-miR863-5p.2–AT1G76550.1, ath-miR822.2–AT5G03552.1, ath-miR822.3–AT5G02350.1, sRNA4–AT1G66290.1 and sRNA6–AT1G66310.1) were identified, providing a basis for in-depth functional analysis of these miRNA candidates. These results could update the current understanding of the biogenesis and the action of the plant miRNAs.

Introduction

MicroRNAs (miRNAs), a well-known small RNA (sRNA) species of ~ 21 nucleotides (nt) in length, play essential regulatory roles in gene expression in a vast range of organisms (Carthew and Sontheimer, 2009, Chen, 2009, Jones-Rhoades et al., 2006). From the primary microRNA (pri-miRNA) to the precursor microRNA (pre-miRNA), then to the miRNA/miRNA* duplex, Dicer-like 1 (DCL1)-mediated two-step cleavages are required for plant miRNA processing. The short duplexes are then methylated and exported to the cytoplasm, in which the guide strand, i.e. miRNA, is incorporated into a specific Argonaute (AGO, normally AGO1) protein to carry out the silencing reaction (Voinnet, 2009). In contrast to the animals', the plant miRNAs bind to the target transcripts harboring highly complementary recognition sites, which serves as a general feature of miRNA-guided cleavage actions in plants (Voinnet, 2009). miRNA*, also defined as the passenger strand, is subjected to rapid degradation, which leads to a much lower accumulation level compared to that of the miRNA (Jones-Rhoades et al., 2006, Voinnet, 2009). However, there are some exceptional cases that miRNA*s are abundant and can interact with AGO proteins to exert their repressive regulatory roles (Okamura et al., 2009, Shao et al., 2013). If the abundances of miRNA and miRNA* are comparable, they are named as miR-5p (on the 5′ arm of the pre-miRNA) and miR-3p (on the 3′ arm of the pre-miRNA) (Ambros et al., 2003, Meyers et al., 2008).

In miRBase (release 19, http://www.mirbase.org/) (Griffiths-Jones et al., 2008), the numbers of registered pre-miRNAs and mature miRNAs of Arabidopsis are 299 and 338, respectively. Statistical result shows that except for ath-MIR1886 which produces 3 miRNAs, a dominant portion of the pre-miRNAs produce only one miRNA (261 out of 299) while only a few of the pre-miRNAs produce two miRNAs (37 out of 299). It is interesting that seven pre-miRNAs (ath-MIR161, ath-MIR447a, ath-MIR779, ath-MIR780, ath-MIR829, ath-MIR869 and ath-MIR1886) in Arabidopsis were annotated to produce more than one miRNA/miRNA* duplexes on their stem regions based on the current miRBase registries (release 19) (Griffiths-Jones et al., 2008).

Using the pathogen infected seedlings, Zhang et al. (2010) reported 19 miRNA precursors in Arabidopsis that can yield multiple distinct miRNA-like sRNAs in addition to the already annotated miRNAs and miRNA*s (Zhang et al., 2010). The biogenesis pathway of these miRNA-like small RNAs is similar to that of the sibling miRNAs, which does not depend on RNA-dependent RNA polymerases or RNA polymerase IV. Some of the miRNA-like sRNAs are differentially expressed in response to bacterial challenges, and some are more abundant than the cognate miRNAs (Zhang et al., 2010).

By considering the organ-specific expression patterns of the miRNAs, it is intriguing to identify some novel miRNA-like sRNA candidates derived from the miRNA precursors in different organs of Arabidopsis.

In this study, by taking advantage of the AGO1-enriched sRNA high-throughput sequencing (HTS) data generated from flower, leaf, root and seedling, we did a comprehensive search for the novel miRNA-like-coding sites on the long-stem pre-miRNAs in Arabidopsis. AGO1 enrichment, co-existence of the miRNA*-like coordinates, and unique genome-wide match sites were taken into consideration for candidate screening. As a result, 43 miRNA-like candidates deriving from 25 pre-miRNAs were identified. Among these candidates, 31 strong candidates from 22 pre-miRNAs passed all the filtering steps, while the other 12 candidates from five pre-miRNAs failed to pass one of the filtering steps. For all the miRNA-like candidates, miRU algorithm-facilitated (Zhang, 2005) target prediction followed by degradome sequencing data-based validation was performed. As a result, five miRNA candidate–target pairs (ath-miR863-5p.2–AT1G76550.1, ath-miR822.2–AT5G03552.1, ath-miR822.3–AT5G02350.1, sRNA4–AT1G66290.1 and sRNA6–AT1G66310.1) were identified, providing a basis for in-depth functional analyses of these novel miRNA-like loci.

Section snippets

Identification of novel miRNA-like candidates derived from long-stem pre-miRNAs in Arabidopsis

In plants, most miRNAs are loaded into AGO1-associated miRNA-induced silencing complexes (miRISCs), guiding target cleavages (Baumberger and Baulcombe, 2005). In this regard, the AGO1-enriched sRNA HTS data were utilized to find the novel miRNA candidates on the long-stem pre-miRNAs. The following criteria were applied: (1) the sRNAs from the AGO1-enriched sequencing data sets that could be perfectly mapped onto the pre-miRNAs were retained; (2) for these sRNAs, the corresponding read counts

Data sources

The sRNA HTS data sets of Arabidopsis were retrieved from GEO (Gene Expression Omnibus; http://www.ncbi.nlm.nih.gov/geo/) (Barrett et al., 2009) and the degradome sequencing data sets were retrieved from GEO and NGSDBs (Next-Gen Sequence Databases; http://mpss.udel.edu/) (Nakano et al., 2006). See Table S1 for the accession numbers and sequencing library information. In order to allow cross-library comparison, the normalized read count (in RPM, reads per million) of a short read from a specific

Competing interests

The authors declare that they have no competing interest.

Acknowledgments

We would like to thank all the publicly available data sets and the scientists behind them. This work was supported by the National Natural Sciences Foundation of China [31271380], [31100937] and [30971743], Scientific Research Fund of Zhejiang Provincial Education Department [Y201222905], Professional Development Projects for the Visiting Scholar of the Colleges and Universities [FX2012055] and the Starting Grant funded by Hangzhou Normal University to Yijun Meng [2011QDL60].

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