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Allelic imbalance sequencing reveals that single-nucleotide polymorphisms frequently alter microRNA-directed repression

Abstract

Genetic changes that help explain the differences between two individuals might create or disrupt sites complementary to microRNAs (miRNAs)1,2, but the extent to which such polymorphic sites influence miRNA-mediated repression is unknown. Here, we describe a method to measure mRNA allelic imbalances associated with a regulatory site found in mRNA transcribed from one allele but not found in that transcribed from the other. Applying this method, called allelic imbalance sequencing, to sites for three miRNAs (miR-1, miR-133 and miR-122) provided quantitative measurements of repression in vivo without altering either the miRNAs or their targets. A substantial fraction of polymorphic sites mediated repression in tissues that expressed the cognate miRNA, and downregulation was correlated with site type and site context. Extrapolating these results to the other broadly conserved miRNAs suggests that when comparing two mouse strains (or two human individuals), polymorphic miRNA sites cause expression of many genes (often hundreds) to differ.

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Figure 1: Measurement of mRNA allelic imbalances associated with heterozygous miRNA target sites.
Figure 2: Impact of heterozygous target sites on mRNA allelic imbalance.
Figure 3: Dependence of target site efficacy on site type and site context.

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Acknowledgements

We thank Tim Harkins and the 454 Sequencing Facility for high-throughput sequencing, members of the laboratory for helpful comments on this manuscript, and T. DiCesare for illustration. Supported by a grant from the National Institutes of Health. D.P.B. is an investigator of the Howard Hughes Medical Institute.

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Contributions

J.K. performed all experiments and analyses. Both authors designed the experiments and wrote the manuscript.

Corresponding author

Correspondence to David P Bartel.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 and Supplementary Discussion (PDF 194 kb)

Supplementary Table 1

Data and information on the SNPs in 3′UTRs that create/disrupt miRNA target sites. (XLS 66 kb)

Supplementary Table 2

Data and information on the SNPs in ORFs. (XLS 23 kb)

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Kim, J., Bartel, D. Allelic imbalance sequencing reveals that single-nucleotide polymorphisms frequently alter microRNA-directed repression. Nat Biotechnol 27, 472–477 (2009). https://doi.org/10.1038/nbt.1540

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