Evolutionary conservation and function of the human embryonic stem cell specific miR-302/367 cluster

Abstract

miRNA clusters define a group of related miRNAs closely localized in the genome with an evolution that remains poorly understood. The miR-302/367 cluster represents a single polycistronic transcript that produces five precursor miRNAs. The cluster is highly expressed and essential for maintenance of human embryonic stem cells. We found the cluster to be highly conserved and present in most mammals. In primates, seed sequence and miRNA structure are conserved, but inter-precursor sequences are evolving. Insertions of new miRNAs, deletions of individual miRNAs, and a cluster duplication observed in different species suggest an actively evolving cluster. Core transcriptional machinery consisting of NANOG and OCT-4 transcription factors that define stem cells are present upstream of the miR-302/367 cluster. Interestingly, we found the miR-302/367 cluster flanking region to be enriched as a target site of other miRNAs suggesting a mechanism for feedback control. Analysis of miR- 302 and miR-367 targets demonstrated concordance of gene set enrichment groups at high gene ontology levels. This cluster also expresses isomiRs providing another means of establishing sequence diversity. Finally, using three different kidney tumor datasets, we observed consistent expression of miR-302 family members in normal tissue while adjacent tumor tissue showed a significant lack of expression. Clustering expression levels of miR- 302 validated target genes showed a significant correlation between miR-302/367 cluster miRNAs and a subset of validated gene targets in healthy and adjacent tumor tissues. Taken together, our data show a highly conserved and still evolving miRNA cluster that may have additional unrecognized functions.