Widespread regulation of miRNA biogenesis at the Dicer step by the cold-inducible RNA-binding protein, RBM3.

Julie Pilotte,Peter W Vanderklish,Esther E Dupont-Versteegden,André Paul Gerber

doi:10.1371/journal.pone.0028446

Julie Pilotte, Peter W Vanderklish + Show 2 more

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https://doi.org/10.1371/journal.pone.0028446

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Journal: PloS one	Publication Date: Dec 1, 2011
Citations: 127	License type: CC BY 4.0

Affiliation: Scripps Research Institute, University of Kentucky

Abstract

MicroRNAs (miRNAs) play critical roles in diverse cellular events through their effects on translation. Emerging data suggest that modulation of miRNA biogenesis at post-transcriptional steps by RNA-binding proteins is a key point of regulatory control over the expression of some miRNAs and the cellular processes they influence. However, the extent and conditions under which the miRNA pathway is amenable to regulation at posttranscriptional steps are poorly understood. Here we show that RBM3, a cold-inducible, developmentally regulated RNA-binding protein and putative protooncogene, is an essential regulator of miRNA biogenesis. Utilizing miRNA array, Northern blot, and PCR methods, we observed that over 60% of miRNAs detectable in a neuronal cell line were significantly downregulated by knockdown of RBM3. Conversely, for select miRNAs assayed by Northern blot, induction of RBM3 by overexpression or mild hypothermia increased their levels. Changes in miRNA expression were accompanied by changes in the levels of their ∼70 nt precursors, whereas primary transcript levels were unaffected. Mechanistic studies revealed that knockdown of RBM3 does not reduce Dicer activity or impede transport of pre-miRNAs into the cytoplasm. Rather, we find that RBM3 binds directly to ∼70 nt pre-miRNA intermediates and promotes / de-represses their ability as larger ribonucleoproteins (pre-miRNPs) to associate with active Dicer complexes. Our findings suggest that the processing of a majority of pre-miRNPs by Dicer is subject to an intrinsic inhibitory influence that is overcome by RBM3 expression. RBM3 may thus orchestrate changes in miRNA expression during hypothermia and other cellular stresses, and in the euthermic contexts of early development, differentiation, and oncogenesis where RBM3 expression is highly elevated. Additionally, our data suggest that temperature-dependent changes in miRNA expression mediated by RBM3 may contribute to the therapeutic effects of hypothermia, and are an important variable to consider in in vitro studies of translation-dependent cellular events.

Highlights

MicroRNAs are a family of short, noncoding RNAs that regulate translation of mRNAs by mechanisms involving the binding of complementary sequences [1,2]
Expression changes identified by microarray were reliably confirmed in Northern blot analyses in which we evaluated the effects of RBM3 overexpression
Our data establish a novel role for RBM3 in the posttranscriptional regulation of miRNA biogenesis, one with important implications for how pre-miRNAs are regulated as RNPs

Summary

Introduction

MicroRNAs (miRNAs) are a family of short, noncoding RNAs that regulate translation of mRNAs by mechanisms involving the binding of complementary sequences [1,2]. Primary transcripts (pri-miRNAs) are first cleaved by Drosha in the nucleus to yield ,70 nt hairpin precursors (pre-miRNAs). These intermediates are transported to the cytoplasm where ,22mer dsRNAs are excised by Dicer. Recent studies indicate that some RNA-binding proteins (RNABPs) can regulate discrete processing steps [3], differentially blocking [14,15] or promoting [16,17,18] the formation of specific miRNAs to control cellular proliferation and differentiation

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