Department of Neurobiology, The Scripps Research Institute, La Jolla, California, United States of America.
PLoS One. 2011;6(12):e28446. doi: 10.1371/journal.pone.0028446. Epub 2011 Dec 1.
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.
微小 RNA（miRNAs）通过对翻译的影响在各种细胞事件中发挥关键作用。新出现的数据表明，RNA 结合蛋白在后转录水平对 miRNA 生物发生的调节是调节某些 miRNA 及其影响的细胞过程表达的关键控制点。然而，miRNA 途径在后转录水平上可调节的程度和条件知之甚少。在这里，我们表明 RBM3，一种冷诱导、发育调节的 RNA 结合蛋白和潜在的原癌基因，是 miRNA 生物发生的重要调节剂。利用 miRNA 阵列、Northern blot 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 miRNAs 被 RBM3 的敲低显著下调。相反，对于通过 Northern blot 测定的选定 miRNAs，通过过表达或轻度低温诱导 RBM3 增加了它们的水平。miRNA 表达的变化伴随着它们的70nt 前体的水平变化，而初级转录物的水平不受影响。机制研究表明，RBM3 的敲低不会降低 Dicer 的活性或阻碍 pre-miRNA 进入细胞质的运输。相反，我们发现 RBM3 直接结合到70nt pre-miRNA 中间体，并促进/解除它们作为更大的核糖核蛋白（pre-miRNPs）与活性 Dicer 复合物结合的能力。我们的发现表明，大多数 pre-miRNPs 的加工受到内在抑制影响，而 RBM3 的表达可以克服这种影响。因此，RBM3 可能在低温和其他细胞应激期间以及在 RBM3 表达高度升高的 euthermic 背景下，如早期发育、分化和癌发生中，协调 miRNA 表达的变化。此外，我们的数据表明，由 RBM3 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，并且在依赖翻译的细胞事件的体外研究中是一个重要的考虑因素。
