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Imperfect centered miRNA binding sites are common and can mediate repression of target mRNAs

Hilary C Martin12, Shivangi Wani15, Anita L Steptoe1, Keerthana Krishnan1, Katia Nones1, Ehsan Nourbakhsh1, Alexander Vlassov3, Sean M Grimmond14 and Nicole Cloonan15*

Author Affiliations

1 Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, Brisbane, St Lucia QLD 4072, Australia

2 Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK

3 Life Technologies, 2130 Woodward St, Austin, TX 78744, USA

4 Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1BD, UK

5 QIMR Berghofer Medical Research Institute, Genomic Biology Laboratory, 300 Herston Road, Herston, QLD 4006, Australia

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Genome Biology 2014, 15:R51  doi:10.1186/gb-2014-15-3-r51

Published: 14 March 2014

Abstract

Background

MicroRNAs (miRNAs) bind to mRNAs and target them for translational inhibition or transcriptional degradation. It is thought that most miRNA-mRNA interactions involve the seed region at the 5′ end of the miRNA. The importance of seed sites is supported by experimental evidence, although there is growing interest in interactions mediated by the central region of the miRNA, termed centered sites. To investigate the prevalence of these interactions, we apply a biotin pull-down method to determine the direct targets of ten human miRNAs, including four isomiRs that share centered sites, but not seeds, with their canonical partner miRNAs.

Results

We confirm that miRNAs and their isomiRs can interact with hundreds of mRNAs, and that imperfect centered sites are common mediators of miRNA-mRNA interactions. We experimentally demonstrate that these sites can repress mRNA activity, typically through translational repression, and are enriched in regions of the transcriptome bound by AGO. Finally, we show that the identification of imperfect centered sites is unlikely to be an artifact of our protocol caused by the biotinylation of the miRNA. However, the fact that there was a slight bias against seed sites in our protocol may have inflated the apparent prevalence of centered site-mediated interactions.

Conclusions

Our results suggest that centered site-mediated interactions are much more frequent than previously thought. This may explain the evolutionary conservation of the central region of miRNAs, and has significant implications for decoding miRNA-regulated genetic networks, and for predicting the functional effect of variants that do not alter protein sequence.