Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014;11(11):1414-29.
doi: 10.1080/15476286.2014.996474.

Comprehensive analysis of small RNA-seq data reveals that combination of miRNA with its isomiRs increase the accuracy of target prediction in Arabidopsis thaliana

Firoz Ahmed  1 , Muthappa Senthil-Kumar, Seonghee Lee, Xinbin Dai, Kirankumar S Mysore, Patrick Xuechun Zhao
Affiliations

Comprehensive analysis of small RNA-seq data reveals that combination of miRNA with its isomiRs increase the accuracy of target prediction in Arabidopsis thaliana

Firoz Ahmed et al. RNA Biol. 2014.

Abstract

Along with the canonical miRNA, distinct miRNA-like sequences called sibling miRNAs (sib-miRs) are generated from the same pre-miRNA. Among them, isomeric sequences featuring slight variations at the terminals, relative to the canonical miRNA, constitute a pool of isomeric sibling miRNAs (isomiRs). Despite the high prevalence of isomiRs in eukaryotes, their features and relevance remain elusive. In this study, we performed a comprehensive analysis of mature precursor miRNA (pre-miRNA) sequences from Arabidopsis to understand their features and regulatory targets. The influence of isomiR terminal heterogeneity in target binding was examined comprehensively. Our comprehensive analyses suggested a novel computational strategy that utilizes miRNA and its isomiRs to enhance the accuracy of their regulatory target prediction in Arabidopsis. A few targets are shared by several members of isomiRs; however, this phenomenon was not typical. Gene Ontology (GO) enrichment analysis showed that commonly targeted mRNAs were enriched for certain GO terms. Moreover, comparison of these commonly targeted genes with validated targets from published data demonstrated that the validated targets are bound by most isomiRs and not only the canonical miRNA. Furthermore, the biological role of isomiRs in target cleavage was supported by degradome data. Incorporating this finding, we predicted potential target genes of several miRNAs and confirmed them by experimental assays. This study proposes a novel strategy to improve the accuracy of predicting miRNA targets through combined use of miRNA with its isomiRs.

Keywords: RNA-Seq; arabidopsis; bioinformatics; isomiRs; miRNA; miRNA target prediction.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Diverse sib-miRs generated from the pre-miRNA ath-MIR156a. The canonical mature miRNA sequence, ath-miR156a, is represented in red. The isomiRs are enclosed into blue lines within 5 nt upstream or downstream of the terminal canonical miRNA. The first column indicates normalized counts as RPM, while the second column lists normalized counts within pre-miRNA (for details, see Materials and Methods).
Figure 2.
Figure 2.
Effect of terminal heterogeneity of ath-miR172a-derived isomiRs on target mRNAs. Green and black in the heatmap indicates the presence and absence of the isomiR target, respectively. The canonical miRNA, ath-miR172a, is denoted with a red box while experimentally validated targets are written in red bold.
Figure 3.
Figure 3.
Position-specific binding of ath-miR172a isomiRs on (A) AT2G28550.1 and (B) AT5G60120.1. Numbering on the x-axis denotes the nucleotide position on the transcript while the y-axis denotes the isomiR. isomiR sequences are provided in Table 3. The numbering of isomiRs on the y-axis corresponds to Serial Number (SN) in Table 3. The canonical miRNA, ath-miR172a, is denoted in red.
Figure 4.
Figure 4.
The interactive Gene Ontology graph of biological processes.
Figure 5.
Figure 5.
Quantification of endogenous mature ath-miR158a and an isomiR158. Total RNA was isolated from 5- week-old Arabidopsis mutant (SALK_025691 and SALK_083227) and wild-type (Col-0) plant leaves. Levels of miR158 and an isomiR158 were quantified using RT-qPCR. Values represented are relative gene expression over 2 internal controls, miR159 and UBQ5. Error bars represent the standard deviation. Asterisks indicate values that were considered statistically significant (p < 0.05) compared to wild-type according to the Student's t-test. Experiment was performed with 2 biological and 3 technical replications.
Figure 6.
Figure 6.
Quantification of endogenous levels of mRNAs predicted to be targeted by ath-miR158a. (A) Expression levels of 4 target mRNAs of ath-miR158a (At1g49910, At1g64100, At3g03580, and At1g62860) were determined in total RNA isolated from Arabidopsis mutant and wild-type leaves. (B) Expression levels of 4 target mRNAs of ath-miR158a (At1g49910, At1g64100, At3g03580, and At1g62860) were determined in total RNA isolated from Arabidopsis mutant and wild-type flowers. Error bars represent the standard deviation. Asterisks indicate values were considered statistically significant (p < 0.05) compared to wild-type according to the Student's t-test.
Figure 7.
Figure 7.
Schematic depicting how miRNA and its isomiRs increase the specificity and efficacy of gene silencing. (A) Each miRNA/isomiR targeting to different group of mRNAs is illustrated as 4 different ovals. Each group contains different genes, as indicated by different symbols within the oval. However, due to high sequence similarity between isomiRs and the miRNA, they also have some common (bona fide) targets. Generally, the abundance of the miRNA is higher than its isomiRs, thus providing a greater degree of silencing, as indicated by the intensity of color in the oval. (B) The cytoplasm contains a myriad of mRNAs. Expression of miRNA and its isomiRs in the cell increases their chance of binding to common “bona fide” targets (star symbol) with a low chance of binding to off-targets. This leads to a greater silencing effect of bona fide target genes and a reduced silencing effect for off-targets, as depicted by the intensity of color in the oval. Common target mRNAs are enriched for Gene Ontology terms.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ul Hussain M. Micro-RNAs (miRNAs): genomic organisation, biogenesis and mode of action. Cell Tissue Res 2012; 349:405-13; PMID:22622804; http://dx.doi.org/10.1007/s00441-012-1438-0 - DOI - PubMed
    1. Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 2009; 10:126-39; PMID:19165215; http://dx.doi.org/10.1038/nrm2632 - DOI - PubMed
    1. Meng Y, Shao C, Wang H, Chen M. The regulatory activities of plant microRNAs: a more dynamic perspective. Plant Physiol 2011; 157:1583-95; PMID:22003084; http://dx.doi.org/10.1104/pp.111.187088 - DOI - PMC - PubMed
    1. Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. Cell 2009; 136:642-55; PMID:19239886; http://dx.doi.org/10.1016/j.cell.2009.01.035 - DOI - PMC - PubMed
    1. Chen X. Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol 2009; 25:21-44; PMID:19575669; http://dx.doi.org/10.1146/annurev.cellbio.042308.113417 - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources