Loop-miRs: active microRNAs generated from single-stranded loop regions

Abstract

MicroRNAs (miRNAs) are key mediators of post-transcriptional gene regulation. The miRNA precursors are processed by the endonucleases Drosha and Dicer into a duplex, bound to an Argonaute protein and unwound into two single-stranded miRNAs. Although alternative ways to generate miRNAs have been discovered, e.g. pre-miRNA cleavage by Ago2 or cleavage products of snoRNAs or tRNAs, all known pathways converge on a double-stranded RNA duplex. Exogenous single-stranded siRNAs (ss-siRNAs) can elicit an effective RNA interference reaction; recent studies have identified chemical modifications increasing their stability and activity. Here, we provide first evidence that endogenous, unmodified, single-stranded RNA sequences are generated from single-stranded loop regions of human pre-miRNA hairpins, the so called loop-miRs. Luciferase assays and immunoprecipitation validate loop-miR activity and incorporation into RNA-induced silencing complexes. This study identifies endogenous miRNAs that are generated from single-stranded regions; hence, it provides evidence that precursor-miRNAs can give rise to three distinct endogenous miRNAs: the guide strand, the passenger strand and the loop-miR.

miRNA Nomenclature: A View Incorporating Genetic Origins, Biosynthetic Pathways, and Sequence Variants

“…Because isomiRs contain different sequences, they may have different targets and thus different cell functions [19]. Great sequencing depth and careful data mining also reveal moRs (microRNA-offset RNAs) [20–22] and single strand RNA fragments released from the loop RNA portion of the precursor miRNA hairpin [23,24] (which we call “loop-origin miRNAs”, or “loRs”). Although functional evidence remains scarce, these variants appear to be independently regulated and may play cooperative, complementary, or alternative roles in cell function and gene regulation compared to their associated miRNAs.…”

“…Small RNA sequencing libraries also identify sequences that derive from the loop of pre-MiRNAs and, like moRs, their relative quantities can vary according to tissue or developmental stage [23,24], suggesting independent regulation of expression and thus independent cell functions. Furthermore, recent studies reported the expression and incorporation of loop-derived short sequences into the RISC and repression of translation of targeted mRNA in in vitro luciferase assays [23,24].…”

Functional small RNAs are generated from select miRNA hairpin loops in flies and mammals

“…While this article was in production, another study (Winter et al 2013) came to our attention describing functional miRNA loop species in human cells.…”

Argonaute-3 activates the let-7a passenger strand microRNA

“…However, highly conserved passenger strands 5 , 6 can also be incorporated into RISCs and serve as active miRNAs bound to Argonaute (Ago) proteins. Additionally, the loop region can give rise to a loop-miR 7 , 8 . For some miRNAs, both arms of the pre-miRNA hairpin are found in RISCs in significant amounts underlining the functional relevance of these less abundant products of miRNA biogenesis 9 .…”