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MicroRNAs and their isomiRs function cooperatively to target common biological pathways

Nicole Cloonan1*, Shivangi Wani1, Qinying Xu1, Jian Gu2, Kristi Lea2, Sheila Heater2, Catalin Barbacioru3, Anita L Steptoe1, Hilary C Martin1, Ehsan Nourbakhsh1, Keerthana Krishnan1, Brooke Gardiner1, Xiaohui Wang3, Katia Nones1, Jason A Steen1, Nicholas A Matigian1, David L Wood1, Karin S Kassahn1, Nic Waddell1, Jill Shepherd1, Clarence Lee4, Jeff Ichikawa4, Kevin McKernan4, Kelli Bramlett2, Scott Kuersten25* and Sean M Grimmond1*

Author Affiliations

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

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

3 Life Technologies, 800 Lincoln Centre Dr., Foster City, CA 94404, USA

4 Life Technologies, 500 Cummings Center, Suite 2400, Beverly, MA 01915, USA

5 Epicentre (An Illumina Company), 726 Post Rd, Madison, WI 53713, USA

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Genome Biology 2011, 12:R126  doi:10.1186/gb-2011-12-12-r126

Published: 30 December 2011

Abstract

Background

Variants of microRNAs (miRNAs), called isomiRs, are commonly reported in deep-sequencing studies; however, the functional significance of these variants remains controversial. Observational studies show that isomiR patterns are non-random, hinting that these molecules could be regulated and therefore functional, although no conclusive biological role has been demonstrated for these molecules.

Results

To assess the biological relevance of isomiRs, we have performed ultra-deep miRNA-seq on ten adult human tissues, and created an analysis pipeline called miRNA-MATE to align, annotate, and analyze miRNAs and their isomiRs. We find that isomiRs share sequence and expression characteristics with canonical miRNAs, and are generally strongly correlated with canonical miRNA expression. A large proportion of isomiRs potentially derive from AGO2 cleavage independent of Dicer. We isolated polyribosome-associated mRNA, captured the mRNA-bound miRNAs, and found that isomiRs and canonical miRNAs are equally associated with translational machinery. Finally, we transfected cells with biotinylated RNA duplexes encoding isomiRs or their canonical counterparts and directly assayed their mRNA targets. These studies allow us to experimentally determine genome-wide mRNA targets, and these experiments showed substantial overlap in functional mRNA networks suppressed by both canonical miRNAs and their isomiRs.

Conclusions

Together, these results find isomiRs to be biologically relevant and functionally cooperative partners of canonical miRNAs that act coordinately to target pathways of functionally related genes. This work exposes the complexity of the miRNA-transcriptome, and helps explain a major miRNA paradox: how specific regulation of biological processes can occur when the specificity of miRNA targeting is mediated by only 6 to 11 nucleotides.