5′ isomiR variation is of functional and evolutionary importance

Abstract

We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3′ and/or 5′ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5′ differences and in support of this we report that a 5′ isomiR-9–1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5′ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes.

Divergent target recognition by coexpressed 5′-isomiRs of miR-142-3p and selective viral mimicry

“…Thus, miR-223 5 ′ -isomiR expression appears to broaden the overall range of miR-223 targets. The prediction that 5 ′ -isomiR expression can impact miRNA target ranges is further supported by the confirmation of small sets of differentially regulated targets for an aberrant miR-307 5 ′ -isomiR in flies (Fukunaga et al 2012) and for transfected 5 ′ -isomiRs of miR-133a, miR-101, and miR-9 (Humphreys et al 2012;Llorens et al 2013;Tan et al 2014). On the other hand, it has been suggested that 5 ′ -isomiRs have highly overlapping targets (Cloonan et al 2011;Llorens et al 2013) and could therefore act redundantly, to increase the effective miRNA dosage or reduce offtarget effects (Cloonan et al 2011).…”

“…These include whether 5 ′ -isomiR expression is regulated in specific tissues, at different developmental stages, or in disease. Instances of spatial and temporal variation in the relative expression of isomiRs (isomiR switching) have been described (Fukunaga et al 2012;Tan et al 2014). For instance, the relative expression of the convergent miR-101 and miR-101+1 isomiRs changes from 1:2 in the brain to 3:1 in fetal lung fibroblasts (Tan et al 2014).…”

“…Instances of spatial and temporal variation in the relative expression of isomiRs (isomiR switching) have been described (Fukunaga et al 2012;Tan et al 2014). For instance, the relative expression of the convergent miR-101 and miR-101+1 isomiRs changes from 1:2 in the brain to 3:1 in fetal lung fibroblasts (Tan et al 2014). Similarly, the expression of the divergent 5 ′ -isomiRs miR-140-3p and miR-140-3p-1 change from ∼6:1 in the nasal epithelium and mucosa to 1:1.6 in the heart.…”

“…′ -isomiR expression appears to be evolutionarily conserved and 5 ′ -isomiRs associate with Ago proteins (Azuma-Mukai et al 2008;Lee et al 2010;Berezikov et al 2011;Cloonan et al 2011;Tan et al 2014;Xia and Zhang 2014). Evidence that endogenously expressed 5…”

MECHANISMS IN ENDOCRINOLOGY: MicroRNA in diagnostics and therapy of thyroid cancer

“…This fact is of profound importance for miRNA's function, because the addition or deletion of nucleotides at the isomiR 5 0 end results in a change in the seed region and, consequently, leads to the regulation of a distinct set of target genes compared to the isomiR's canonical counterpart. As functional partners of their reference canonical molecules, isomiRs are tissue specific; therefore, the proper interpretation of microRNA-regulated networks requires comprehensive information on the miRNome for each particular tissue type (15,16,17).…”