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. 2013 Apr;161(4):1875-84.
doi: 10.1104/pp.113.215962. Epub 2013 Feb 21.

Widespread long noncoding RNAs as endogenous target mimics for microRNAs in plants

Hua-Jun Wu  1 , Zhi-Min Wang, Meng Wang, Xiu-Jie Wang
Affiliations

Widespread long noncoding RNAs as endogenous target mimics for microRNAs in plants

Hua-Jun Wu et al. Plant Physiol. 2013 Apr.

Abstract

Target mimicry is a recently identified regulatory mechanism for microRNA (miRNA) functions in plants in which the decoy RNAs bind to miRNAs via complementary sequences and therefore block the interaction between miRNAs and their authentic targets. Both endogenous decoy RNAs (miRNA target mimics) and engineered artificial RNAs can induce target mimicry effects. Yet until now, only the Induced by Phosphate Starvation1 RNA has been proven to be a functional endogenous microRNA target mimic (eTM). In this work, we developed a computational method and systematically identified intergenic or noncoding gene-originated eTMs for 20 conserved miRNAs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). The predicted miRNA binding sites were well conserved among eTMs of the same miRNA, whereas sequences outside of the binding sites varied a lot. We proved that the eTMs of miR160 and miR166 are functional target mimics and identified their roles in the regulation of plant development. The effectiveness of eTMs for three other miRNAs was also confirmed by transient agroinfiltration assay.

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Figures

Figure 1.
Figure 1.
Conservation of TM sites among predicted miRNA eTMs in Arabidopsis and rice. A, Box plot for the conservation status of TM sites and their surrounding sequences. The y axis represents the occurrence rate of the most frequently occurred nucleotide at each position among all eTMs in Arabidopsis and rice. The x axis represents nucleotide positions in eTMs. Boxes represent the interquartile range (including the 25th percentile to the 75th percentile of the data) of the most frequent nucleotide occurrence rates of all predicted eTMs at each position. TM sites pairing to miRNAs are represented by blue boxes, and TM sites surrounding sequences are represented by orange boxes. Black lines within boxes represent the median of each data set. Vertical dashed lines represent the range of each data set. Extreme values within each data set are represented by circles. B, Conservation analysis of the TM sites and surrounding regions of 13 eTMs for miR160 in Arabidopsis and rice. TM sites pairing with miRNAs are underlined by blue boxes, and the bulge sequences in eTMs are underlined by red boxes. The conservation status of the sequences is analyzed and presented by WebLogo.
Figure 2.
Figure 2.
Expression of predicted eTMs in Arabidopsis and rice. A, Expression profile of predicted eTMs in flower, seedling, and root tissues of Arabidopsis obtained from public RNA-Seq data (SRP007511). B, Expression profile of predicted eTMs in nine tissues of rice obtained from public RNA-Seq data (SRP008821). For both A and B, expression is presented by the reads per kilobase per million mapped reads values of transcripts corresponding to each eTM after log2 transformation. Gray color represents no expression in RNA-Seq data. C, Semiquantitative RT-PCR assay of eTMs in eight Arabidopsis samples. Samples were collected from 2-week-old seedlings (seedling root and seedling leaf) and 5-week-old plants. D, Semiquantitative RT-PCR assay of eTMs in four rice samples. Samples were collected from heading stage (flag leaves and flowers) and seedling stage (3-week-old seedling root and 3-week-old seedling leaf) rice. RT- represents the RT-minus negative control (without the addition of reverse transcriptase).
Figure 3.
Figure 3.
Functional analysis of ath-eTM160-1. A, Predicted base-pairing interaction between miR160 and ath-eTM160-1. B, Detection of enhanced eTM expression in wild-type (WT) and ath-eTM160-1 overexpression transgenic Arabidopsis by semiquantitative RT-PCR assay. OE-ath-eTM160-1(1) and OE-ath-eTM160-1(10) represent two independent transgenic lines overexpressing ath-eTM160-1. C, Phenotypes of ath-eTM160-1 overexpression plants. Smaller leaf size and serrated leaf margin are observed. D, Quantitative RT-PCR analysis of miR160 target genes (ARF10, ARF16, and ARF17) in wild-type and OE-ath-eTM160-1 plants. Error bars indicate the sd of three replicates. E, Detection of miR160 expression in OE-ath-eTM160-1 plants by northern-blot hybridization. rRNA, Ribosomal RNA.
Figure 4.
Figure 4.
Functional analysis of osa-eTM160-3. A, Predicted base-pairing interaction between miR160 and osa-eTM160-3 (top alignment) as well as miR160 and osa-eTM160-3 with designed mutations (osa-eTM160-3M; bottom alignment). Mutation sites are shown in red. B, Detection of enhanced eTM expression by semiquantitative RT-PCR assay in wild-type Arabidopsis (WT) and Arabidopsis overexpressing osa-eTM160-3 (OE-osa-eTM160-3) as well as plants overexpressing osa-eTM160-3 with designed mutations (OE-osa-eTM160-3M). C, Phenotypes of osa-eTM160-3 and osa-eTM160-3M overexpression plants. Smaller leaf size and serrated leaf margin are observed in OE-osa-eTM160-3 plants, yet OE-osa-eTM160-3M plants look normal. D, Quantitative RT-PCR analysis of miR160 target genes (ARF10, ARF16, and ARF17) in wild-type and OE-osa-eTM160-3 as well as OE-osa-eTM160-3M plants. Error bars indicate the sd of three replicates. The increased miR160 target expression in OE-osa-eTM160-3 plants is abolished in OE-osa-eTM160-3M plants. E, Detection of miR160 expression in OE-osa-eTM160-3 and OE-osa-eTM160-3M plants by northern-blot hybridization. rRNA, Ribosomal RNA.
Figure 5.
Figure 5.
Overexpression of ath-miR160c rescues the phenotypes of OE-osa-eTM160-3. A, Phenotype analysis of plants overexpressing both osa-eTM160-3 and ath-miR160c. OE-ath-miR160c represents transgenic plants overexpressing ath-miR160c, which can pair with osa-eTM160-3. WT, Wild type. B, Expression analysis of miR160 targets in transgenic plants overexpressing both osa-eTM160-3 and ath-miR160c by quantitative RT-PCR. The expression of three miR160 target genes (ARF10, ARF16, and ARF17) was tested in three independent lines (labeled 1, 2, and 3). Error bars indicate the sd of three replicates.
Figure 6.
Figure 6.
Functional analysis of ath-eTM166-1. A, Predicted base-pairing interaction between miR166 and ath-eTM166-1. B, Phenotypes of ath-eTM166-1 overexpression plants. Spoon-shaped cotyledons and curled rosette leaves are observed. C, Quantitative RT-PCR analysis of miR166 target genes (ATHB9, ATHB14, and ATHB15) in wild-type (WT) and OE-ath-eTM166-1 plants. OE-ath-eTM166-1(4) and OE-ath-eTM166-1(6) represent two independent transgenic lines overexpressing ath-eTM166-1. Error bars indicate the SD of three replicates.
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