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Global identification of microRNA–target RNA pairs by parallel analysis of RNA ends

Nature Biotechnology volume 26, pages 941–946 (2008)Cite this article

Abstract

MicroRNAs (miRNAs) are important regulatory molecules in most eukaryotes and identification of their target mRNAs is essential for their functional analysis. Whereas conventional methods rely on computational prediction and subsequent experimental validation of target RNAs, we directly sequenced >28,000,000 signatures from the 5′ ends of polyadenylated products of miRNA-mediated mRNA decay, isolated from inflorescence tissue of Arabidopsis thaliana, to discover novel miRNA–target RNA pairs. Within the set of ∼27,000 transcripts included in the 8,000,000 nonredundant signatures, several previously predicted but nonvalidated targets of miRNAs were found. Like validated targets, most showed a single abundant signature at the miRNA cleavage site, particularly in libraries from a mutant deficient in the 5′-to-3′ exonuclease AtXRN4. Although miRNAs in Arabidopsis have been extensively investigated, working in reverse from the cleaved targets resulted in the identification and validation of novel miRNAs. This versatile approach will affect the study of other aspects of RNA processing beyond miRNA–target RNA pairs.

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Figure 1: Target plots (t-plots) of validated target mRNAs.
Figure 2: Novel targets of miRNAs were recognized based on their t-plots.
Figure 3: Pri-miRNAs having signatures corresponding to the cleavage site of their own mature miRNAs.

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Acknowledgements

We are grateful to Mayumi Nakano for the web interface, Shawn Thatcher for help in editing the manuscript and members of the Green and Meyers Lab for helpful discussions. This work was supported primarily by Department of Energy no. DE-FG02-04ER15541 and National Science Foundation (NSF) no. 0445638 (P.J.G.), with additional support from US Department of Agriculture no. 2007-01991 (P.J.G.), NSF #0548569 (P.J.G and B.C.M.), NSF #0321437 (B.C.M.) and National Institutes of Health P20 RR16472-04.

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Authors and Affiliations

  1. Delaware Biotechnology Institute, University of Delaware, Newark, 19711, Delaware, USA

    Marcelo A German, Manoj Pillay, Dong-Hoon Jeong, Amit Hetawal, Prakash Janardhanan, Vimal Kannan, Linda A Rymarquis, Kan Nobuta, Rana German, Emanuele De Paoli, Cheng Lu, Blake C Meyers & Pamela J Green

  2. Illumina, Inc., 25861 Industrial Blvd., Hayward, 94545, California, USA

    Shujun Luo & Gary Schroth

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  1. Marcelo A German
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Contributions

M.A.G. conceived and initiated PARE research, designed and carried out experimental work, analyzed the data and wrote the paper, D.-H.J., S.L., R.G. and C.L. carried out experimental work, M.P. and K.N. performed and supervised computational work, A.H., P.J., V.K., L.A.R. and E.D.P. performed computational work, G.S. supervised experimental work, B.C.M. coordinated and supervised computational work and wrote the paper, P.J.G. designed and supervised experimental work and wrote the paper.

Corresponding authors

Correspondence to Blake C Meyers or Pamela J Green.

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Competing interests

G.S., S.L. and K.N. are employees of Illumina, which is the company that has commercialized the SBS sequencing technology. Our paper describes the use of this technology for the analysis of 5′ RNA ends; we demonstrate this approach in Arabidopsis, but the method has broad applicability.

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Figures 1–7, Tables 1–6 (PDF 1857 kb)

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German, M., Pillay, M., Jeong, DH. et al. Global identification of microRNA–target RNA pairs by parallel analysis of RNA ends. Nat Biotechnol 26, 941–946 (2008). https://doi.org/10.1038/nbt1417

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