A novel role for GSK3β as a modulator of Drosha microprocessor activity and MicroRNA biogenesis.

Claire E Fletcher,Akifumi Shibakawa,Martin Bushell,Jack D Godfrey,Charlotte L Bevan

doi:10.1093/nar/gkw938

Claire E Fletcher, Akifumi Shibakawa + Show 3 more

Open Access

https://doi.org/10.1093/nar/gkw938

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Abstract

Regulation of microRNA (miR) biogenesis is complex and stringently controlled. Here, we identify the kinase GSK3β as an important modulator of miR biogenesis at Microprocessor level. Repression of GSK3β activity reduces Drosha activity toward pri-miRs, leading to accumulation of unprocessed pri-miRs and reduction of pre-miRs and mature miRs without altering levels or cellular localisation of miR biogenesis proteins. Conversely, GSK3β activation increases Drosha activity and mature miR accumulation. GSK3β achieves this through promoting Drosha:cofactor and Drosha:pri-miR interactions: it binds to DGCR8 and p72 in the Microprocessor, an effect dependent upon presence of RNA. Indeed, GSK3β itself can immunoprecipitate pri-miRs, suggesting possible RNA-binding capacity. Kinase assays identify the mechanism for GSK3β-enhanced Drosha activity, which requires GSK3β nuclear localisation, as phosphorylation of Drosha at S300 and/or S302; confirmed by enhanced Drosha activity and association with cofactors, and increased abundance of mature miRs in the presence of phospho-mimic Drosha. Functional implications of GSK3β-enhanced miR biogenesis are illustrated by increased levels of GSK3β-upregulated miR targets following GSK3β inhibition. These data, the first to link GSK3β with the miR cascade in humans, highlight a novel pro-biogenesis role for GSK3β in increasing miR biogenesis as a component of the Microprocessor complex with wide-ranging functional consequences.

Highlights

MicroRNAs, first identified in 1993, are 18–22 nucleotide non-coding RNAs
As it has been previously demonstrated that GSK3␤ can phosphorylate Drosha and alter its subcellular localisation [34,41], it was possible that the effects of GSK3␤ inhibition on miR maturation may be attributable to altered Drosha localisation
GSK3␤ has been shown to phosphorylate Drosha at residues S300 and S302, and it has been suggested that such modifications are required for Drosha nuclear localisation [34]

Summary

Introduction

MicroRNAs, first identified in 1993, are 18–22 nucleotide non-coding RNAs. The accepted dogma is that they negatively regulate gene expression through association with complementary sequences within target gene 3 UTRs, leading to transcript degradation and/or translational inhibition [1,2]. An RNase III enzyme, is stabilised by association with double-stranded RNA binding domain protein DiGeorge Critical Region 8 (DGCR8)/Partner of Drosha (Pasha) [7]. Other cofactors such as p72, p68, FUS and hnRNPA1 modulate fidelity, efficiency and specificity of cleavage or act as scaffold proteins to aid complex formation [8]. The mature miR guides RISC to complementary sequences within the 3 UTR of target mRNAs, resulting in translational repression and/or transcript degradation

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