Background

Brassinosteroids (BRs) are a class of phytohormones with important roles in regulating physiological and developmental processes. Small RNAs, including small interfering RNAs and microRNAs (miRNAs), are non-protein coding RNAs that regulate gene expression at the transcriptional and post-transcriptional levels. However, the roles of small RNAs in BR response have not been studied well.

Objective

In this study, we aimed to identify BR-responsive small RNA clusters and miRNAs in Arabidopsis. In addition, the effect of BR-responsive small RNAs on their transcripts and target genes were examined.

Methods

Small RNA libraries were constructed from control and epibrassinolide-treated seedlings expressing wild-type BRI1-Flag protein under its native promoter in the bri1-5 mutant. After sequencing the small RNA libraries, differentially expressed small RNA clusters were identified by examining the expression levels of small RNAs in 100-nt bins of the Arabidopsis genome. To identify the BR-responsive miRNAs, the expression levels of all the annotated mature miRNAs, registered in miRBase, were analyzed. Previously published RNA-seq data were utilized to monitor the BR-responsive expression patterns of differentially expressed small RNA clusters and miRNA target genes.

Results

In results, 38 BR-responsive small RNA clusters, including 30 down-regulated and eight up-regulated clusters, were identified. These differentially expressed small RNA clusters were from miRNA loci, transposons, protein-coding genes, pseudogenes and others. Of these, a transgene, BRI1, accumulates small RNAs, which are not found in the wild type. Small RNAs in this transgene are up-regulated by BRs while BRI1 mRNA is down-regulated by BRs. By analyzing the expression patterns of mature miRNAs, we have identified BR-repressed miR398a-5p and BR-induced miR156g. Although miR398a-5p is down-regulated by BRs, its predicted targets were not responsive to BRs. However, SPL3, a target of BR-inducible miR156g, is down-regulated by BRs.

Conclusion

BR-responsive small RNAs and miRNAs identified in this study will provide an insight into the role of small RNAs in BR responses in plants. Especially, we suggest that miR156g/SPL3 module might play a role in BR-mediated growth and development in Arabidopsis.

中文翻译：

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全基因组分析拟南芥中油菜素类固醇反应性小RNA。

背景

油菜素类固醇（BRs）是一类植物激素，在调节生理和发育过程中具有重要作用。小RNA，包括小干扰RNA和microRNA（miRNA），是非蛋白质编码RNA，可在转录和转录后水平调节基因表达。然而，小RNA在BR反应中的作用尚未得到很好的研究。

目的

在这项研究中，我们旨在鉴定拟南芥中BR反应性小RNA簇和miRNA。另外，检查了BR应答性小RNA对其转录物和靶基因的影响。

方法

由对照和表油菜素内酯处理的幼苗在bri1-5突变体的天然启动子下表达野生型BRI1-Flag蛋白，​​构建了小RNA库。对小RNA文库进行测序后，通过检查拟南芥基因组100 nt仓中小RNA的表达水平，鉴定出差异表达的小RNA簇。为了鉴定BR反应性miRNA，分析了在miRBase中注册的所有带注释的成熟miRNA的表达水平。先前发布的RNA-seq数据用于监测差异表达的小RNA簇和miRNA靶基因的BR反应表达模式。

结果

结果，鉴定出38个BR反应性小RNA簇，包括30个下调的簇和8个上调的簇。这些差异表达的小RNA簇来自miRNA基因座，转座子，蛋白质编码基因，假基因等。其中，转基因BRI1会积聚小分子RNA，这在野生型中不存在。此转基因中的小RNA被BR上调，而BRI1 mRNA被BR上调。通过分析成熟miRNA的表达模式，我们确定了BR抑制的miR398a-5p和BR诱导的miR156g。尽管miR398a-5p受到BR的下调，但其预测的靶标对BR没有反应。然而，SPL3通过生物圈，BR诱导miR156g的目标，是下调。

结论

在这项研究中鉴定出的BR反应性小RNA和miRNA将提供小RNA在植物BR反应中的作用的见解。特别是，我们建议miR156g / SPL3模块可能在拟南芥中BR介导的生长发育中起作用。