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

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.

The RNA binding protein EWS is broadly involved in the regulation of pri-miRNA processing in mammalian cells

“…The Microprocessor Drosha/DGCR8 is responsible for releasing individual pre-miRNAs from pri-miRNA transcripts; upon export to the cytoplasm, pre-miRNAs are further processed into mature miRNAs by Dicer/TRBP, which are finally incorporated into the RNA-induced silencing complex (RISC) for functional execution on target mRNAs ( 24 ). Importantly, each of these processing steps has been shown to subject to modulation by various RBPs ( 24 ) as well as by post-translational modification of the core machineries ( 51 , 52 ). Relevant to our current study, multiple RBPs have been implicated in the regulation of the Microprocessor-mediated conversion from pri-miRNA to pre-miRNA, including positive regulators, such as DDX5/17 (aka p68/p72) ( 53 – 56 ), hnRNP A1 ( 57 , 58 ), and KSRP ( 59 ), and negative regulators, such as Lin28 ( 60 ) and ADAR1/2 ( 61 , 62 ).…”

A pan-cancer atlas of somatic mutations in miRNA biogenesis genes

“…Facilitates pri-miRNA binding by DROSHA and enhances DROSHA association with cofactors DGCR8 and P72 [136]; DROSHA phosphorylation/stabilization [137]…”

Regulation of primary microRNA processing

“…Within the two main modes of action on either the Microprocessor or the pri‐miRNA, several distinct mechanisms can be distinguished to more precisely explain how an accessory protein can impact on miRNA processing. Namely, for proteins directly binding the Microprocessor, we can distinguish between Microprocessor post‐translational modification and direct binding competition between Drosha and DGCR8 . Proteins interacting with the pri‐miRNA can either act by regulating its binding by the Microprocessor either negatively or positively , by remodeling the transcript structure , or by inducing pri‐miRNA post‐transcriptional modifications .…”

“…For example, Drosha is phosphorylated by the protein kinase Glycogen synthase kinase 3β (GSK3β), implicated also in a large number of signaling pathways involving proteins such as Hedgehog, Notch, and WNT/β‐catenin. Interestingly, GSK3β is only able to act in an RNA‐dependent manner, since it cannot directly bind Drosha or DGCR8 . Moreover, at least 23 phosphorylated amino acids have been described on DGCR8.…”