Analyses of a Glycine max Degradome Library Identify microRNA Targets and MicroRNAs that Trigger Secondary SiRNA Biogenesis

Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs. In this study, a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced. 428 potential targets of small interfering RNAs and 25 novel miRNA families were identified. A total of 211 potential miRNA targets, including 174 conserved miRNA targets and 37 soybean-specific miRNA targets, were identified. Among them, 121 targets were first discovered in soybean. The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing. The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean, and nine Auxin Response Factors were identified as TAS3 siRNA targets. Twenty-three miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean. These targets were guided by five miRNAs: gma-miR393, gma-miR1508, gma-miR1510, gma-miR1514, and novel-11. Multiple targets of these secondary siRNAs were detected. These 23 miRNA targets may be the putative novel TAS genes in soybean. Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.     

Common functions for diverse small RNAs of land plants

Endogenous small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), are critical components of plant gene regulation. Some abundant miRNAs involved in developmental control are conserved between anciently diverged plants, while many other less-abundant miRNAs appear to have recently emerged in the Arabidopsis thaliana lineage. Using large-scale sequencing of small RNAs, we extended the known diversity of miRNAs in basal plants to include 88 confidently annotated miRNA families in the moss Physcomitrella patens and 44 in the lycopod Selaginella moellendorffii. Cleavage of 29 targets directed by 14 distinct P. patens miRNA families and a trans-acting siRNA (ta-siRNA) was experimentally confirmed. Despite a core set of 12 miRNA families also expressed in angiosperms, weakly expressed and apparently lineage-specific miRNAs accounted for the majority of miRNA diversity in both species. Nevertheless, the molecular functions of several of these lineage-specific small RNAs matched those of angiosperms, despite dissimilarities in the small RNA sequences themselves, including small RNAs that mediated negative feedback regulation of the miRNA pathway and miR390-dependent ta-siRNAs that guided the cleavage of AUXIN RESPONSE FACTOR mRNAs. Diverse, lineage-specific, small RNAs can therefore perform common biological functions in plants.     

Identification of novel microRNAs in rice (Oryza sativa) based on the cleavage signals in precursors

MicroRNAs (miRNAs) are crucial regulators of gene expression in plants and a growing number of novel miRNA genes have been cloned in rice in recent years. However, there is no evidence that all miRNAs have been discovered, especially for those low expression ones which are difficult to be found by conventional methods. By taking advantage of the finding that DCL1-mediated cleavage signals for the processing of the miRNA precursors could be used as the clues for novel miRNAs’ discovery, a genome-wide search for rice miRNA candidates was carried out. As a result, 51 previously validated miRNAs and 24 novel miRNA candidates were discovered. After target prediction and degradome sequencing data-based validation, coupled with reverse approach retest, 10 miRNA candidate–mRNA target pairs were further identified, providing a basis for in-depth functional analysis of these miRNA candidates. Besides, some isomiRs found in this study showed more likely to be the real miRNAs. We also found an exceptional example which did not obey the rule that 22-nt miRNAs have the ability to trigger the phased siRNAs production from the cleaved targets.     

降解组测序技术在植物miRNA研究中的应用

目前, 利用芯片技术和miRNA测序可快速、准确地检测到物种中所含有的miRNA。随着越来越多的miRNA被发现, miRNA靶基因的确定已成为研究miRNA生物学功能的关键。传统的miRNA靶基因的寻找主要依赖生物信息学预测、AGO蛋白免疫共沉淀和荧光素酶法等。随着高通量测序技术的持续革新, 出现了一种新的miRNA靶基因的检测方法, 即降解组测序(degradome sequencing)法, 该方法拥有高通量测序技术、生物信息学分析和RACE验证三者的优势, 并已成功应用于拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)和小立碗藓(Physcomitrella patens)等模式植物miRNA靶基因的检测。基于已发表的相关文献和联川生物降解组测序平台, 该文对降解组测序技术应用于植物miRNA靶基因的研究进展及其实验原理进行了综述, 同时对运用该技术可进行的更深入研究进行了讨论。     

Small RNA and Degradome Sequencing Reveal Complex Roles of miRNAs and Their Targets in Developing Wheat Grains

Plant microRNAs (miRNAs) have been shown to play critical roles in plant development. In this study, we employed small RNA combined with degradome sequencing to survey development-related miRNAs and their validated targets during wheat grain development. A total of 186 known miRNAs and 37 novel miRNAs were identified in four small RNA libraries. Moreover, a miRNA-like long hairpin locus was first identified to produce 21~22-nt phased siRNAs that act in trans to cleave target mRNAs. A comparison of the miRNAomes revealed that 55 miRNA families were differentially expressed during the grain development. Predicted and validated targets of these development-related miRNAs are involved in different cellular responses and metabolic processes including cell proliferation, auxin signaling, nutrient metabolism and gene expression. This study provides insight into the complex roles of miRNAs and their targets in regulating wheat grain development.     

Transposable element-associated microRNA hairpins produce 21-nt sRNAs integrated into typical microRNA pathways in rice

microRNAs (miRNAs) are a class of small RNAs (sRNAs) of ～21 nucleotides (nt) in length processed from foldback hairpins by DICER-LIKE1 (DCL1) or DCL4. They regulate the expression of target mRNAs by base pairing through RNA-induced silencing complex (RISC). In the RISC, ARGONAUTE1 (AGO1) is the key protein that cleaves miRNA targets at position ten of a miRNA:target duplex. The authenticity of many annotated rice miRNA hairpins is under debate because of their homology to repeat sequences. Some of them, like miR1884b, have been removed from the current release of miRBase based on incomplete information. In this study, we investigated the association of transposable element (TE)-derived miRNAs with typical miRNA pathways (DCL1/4- and AGO1-dependent) using publicly available deep sequencing datasets. Seven miRNA hairpins with 13 unique sRNAs were specifically enriched in AGO1 immunoprecipitation samples and relatively reduced in DCL1/4 knockdown genotypes. Interestingly, these species are ～21-nt long, instead of 24-nt as annotated in miRBase and the literature. Their expression profiles meet current criteria for functional annotation of miRNAs. In addition, diagnostic cleavage tags were found in degradome datasets for predicted target mRNAs. Most of these miRNA hairpins share significant homology with miniature inverted-repeat transposable elements, one type of abundant DNA transposons in rice. Finally, the root-specific production of a 24-nt miRNA-like sRNA was confirmed by RNA blot for a novel EST that maps to the 3′-UTR of a candidate pseudogene showing extensive sequence homology to miR1884b hairpin. Our data are consistent with the hypothesis that TEs can serve as a driving force for the evolution of some MIRNAs, where co-opting of DICER-LIKE1/4 processing and integration into AGO1 could exapt transcribed TE-associated hairpins into typical miRNA pathways.     

SeqTar: an effective method for identifying microRNA guided cleavage sites from degradome of polyadenylated transcripts in plants

In plants, microRNAs (miRNAs) regulate their mRNA targets by precisely guiding cleavages between the 10th and 11th nucleotides in the complementary regions. High-throughput sequencing-based methods, such as PARE or degradome profiling coupled with a computational analysis of the sequencing data, have recently been developed for identifying miRNA targets on a genome-wide scale. The existing algorithms limit the number of mismatches between a miRNA and its targets and strictly do not allow a mismatch or G:U Wobble pair at the position 10 or 11. However, evidences from recent studies suggest that cleavable targets with more mismatches exist indicating that a relaxed criterion can find additional miRNA targets. In order to identify targets including the ones with weak complementarities from degradome data, we developed a computational method called SeqTar that allows more mismatches and critically mismatch or G:U pair at the position 10 or 11. Precisely, two statistics were introduced in SeqTar, one to measure the alignment between miRNA and its target and the other to quantify the abundance of reads at the center of the miRNA complementary site. By applying SeqTar to publicly available degradome data sets from Arabidopsis and rice, we identified a substantial number of novel targets for conserved and non-conserved miRNAs in addition to the reported ones. Furthermore, using RLM 5'-RACE assay, we experimentally verified 12 of the novel miRNA targets (6 each in Arabidopsis and rice), of which some have more than 4 mismatches and have mismatches or G:U pairs at the position 10 or 11 in the miRNA complementary sites. Thus, SeqTar is an effective method for identifying miRNA targets in plants using degradome data sets.     

Genome-Wide Identification of Reverse Complementary microRNA Genes in Plants

MicroRNAs (miRNAs) are ∼21-nucleotide small RNAs (sRNAs) with essential regulatory roles in plants. They are generated from stem-loop-structured precursors through two sequential Dicer-like 1 (DCL1)-mediated cleavages. To date, hundreds of plant miRNAs have been uncovered. However, the question, whether the sequences reverse complementary (RC) to the miRNA precursors could form hairpin-like structures and produce sRNA duplexes similar to the miRNA/miRNA* pairs has not been solved yet. Here, we interrogated this possibility in 16 plant species based on sRNA high-throughput sequencing data and secondary structure prediction. A total of 59 RC sequences with great potential to form stem-loop structures and generate miRNA/miRNA*-like duplexes were identified in ten plants, which were named as RC-miRNA precursors. Unlike the canonical miRNAs, only a few cleavage targets of the RC-miRNAs were identified in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), and none in Soybean (Glycine max) based on degradome data. Surprisingly, the genomic regions surrounding some of the RC-miRNA target recognition sites were observed to be specifically methylated in both Arabidopsis and rice. Taken together, we reported a new class of miRNAs, called RC-miRNAs, which were generated from the antisense strands of the miRNA precursors. Based on the results, we speculated that the mature RC-miRNAs might have subtle regulatory activity through target cleavages, but might possess short interfering RNA-like activity by guiding sequence-specific DNA methylation.     

SRNAome and degradome sequencing analysis reveals specific regulation of sRNA in response to chilling injury in tomato fruit

Plant genomes encode diverse small RNA classes that function in distinct gene‐silencing pathways. To elucidate the intricate regulation of microRNAs (miRNAs) and endogenous small‐interfering RNAs (siRNAs) in response to chilling injury in tomato fruit, the deep sequencing and bioinformatic methods were combined to decipher the small RNAs landscape in the control and chilling‐injured groups. Except for the known miRNAs and ta‐siRNAs, 85 novel miRNAs and 5 ta‐siRNAs members belonging to 3 TAS families (TAS5, TAS9 and TAS10) were identified, 34 putative phased small RNAs and 740 cis/trans‐natural antisense small‐interfering RNAs (nat‐siRNAs) were also found in our results which enriched the tomato small RNAs repository. A large number of genes targeted by those miRNAs and siRNAs were predicted to be involved in the chilling injury responsive process and five of them were verified via degradome sequencing. Based on the above results, a regulatory model that comprehensively reveals the relationships between the small RNAs and their targets was set up. This work provides a foundation for further study of the regulation of miRNAs and siRNAs in the plant in response to chilling injury.     

Integration of expressed sequence tag data flanking predicted RNA secondary structures facilitates novel non-coding RNA discovery

Many computational methods have been used to predict novel non-coding RNAs (ncRNAs), but none, to our knowledge, have explicitly investigated the impact of integrating existing cDNA-based Expressed Sequence Tag (EST) data that flank structural RNA predictions. To determine whether flanking EST data can assist in microRNA (miRNA) prediction, we identified genomic sites encoding putative miRNAs by combining functional RNA predictions with flanking ESTs data in a model consistent with miRNAs undergoing cleavage during maturation. In both human and mouse genomes, we observed that the inclusion of flanking ESTs adjacent to and not overlapping predicted miRNAs significantly improved the performance of various methods of miRNA prediction, including direct high-throughput sequencing of small RNA libraries. We analyzed the expression of hundreds of miRNAs predicted to be expressed during myogenic differentiation using a customized microarray and identified several known and predicted myogenic miRNA hairpins. Our results indicate that integrating ESTs flanking structural RNA predictions improves the quality of cleaved miRNA predictions and suggest that this strategy can be used to predict other non-coding RNAs undergoing cleavage during maturation.