Widespread regulation of miRNA biogenesis at the Dicer step by the cold-inducible RNA-binding protein, RBM3.
Widespread regulation of miRNA biogenesis at the Dicer step by the cold-inducible RNA-binding protein, RBM3.
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DOI：
10.1371/journal.pone.0028446
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Vanderklish PW
中科院分区：
文献类型：
--
作者：
Pilotte J;Dupont-Versteegden EE;Vanderklish PW
microRNAs（miRNAs）通过其对翻译的影响在多种细胞事件中发挥关键作用。新出现的数据表明，通过RNA结合蛋白在转录后步骤调节miRNA生物合成是调控某些miRNA表达及其影响的细胞过程的关键点。然而，在何种程度和条件下的miRNA途径是服从于在转录后步骤的调控知之甚少。在这里，我们表明，RBM 3，冷诱导，发育调节RNA结合蛋白和推定的原癌基因，是一个必不可少的调节器的miRNA生物合成。利用miRNA阵列、北方印迹和PCR方法，我们观察到在神经元细胞系中可检测到的超过60%的miRNA被RBM 3的敲低显著下调。相反，对于通过北方印迹分析的选择的miRNA，通过过表达或轻度低温诱导RBM 3增加了它们的水平。miRNA表达的变化伴随着它们的前体70 nt水平的变化，而初级转录水平不受影响。机制研究表明，RBM 3的敲低不会降低Dicer活性或阻碍pre-miRNA转运到细胞质中。相反，我们发现RBM 3直接结合至约70 nt的前体miRNA中间体，并促进/去抑制它们作为较大的核糖核蛋白（前体miRNP）与活性Dicer复合物缔合的能力。我们的研究结果表明，大多数pre-miRNP的Dicer的处理受到RBM 3表达所克服的内在抑制影响。因此，RBM 3可能在低温和其他细胞应激期间以及在RBM 3表达高度升高的早期发育、分化和肿瘤发生的温温环境中协调miRNA表达的变化。此外，我们的数据表明，RBM 3介导的miRNA表达的温度依赖性变化可能有助于低温的治疗效果，并且是在体外研究抑制依赖性细胞事件中考虑的重要变量。
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.
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