Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030; Cancer Biology Program, The University of Texas Graduate School of Biomedical Sciences, Houston, Texas 77030.
Department of Chemistry and Physics, Western Carolina University, Cullowhee, North Carolina 28723.
J Biol Chem. 2011 Dec 23;286(51):44095-44103. doi: 10.1074/jbc.M111.265439. Epub 2011 Nov 2.
MicroRNAs (miRNA) are endogenous, short, non-coding RNA that undergo a multistep biogenesis before generating the functional, mature sequence. The core components of the microprocessor complex, consisting of Drosha and DGCR8, are both necessary and sufficient for this process, although accessory proteins have been found that modulate the biogenesis of a subset of miRNA. Curiously, many of the proteins involved in miRNA biogenesis are also needed for ribosomal RNA processing. Here we show that nucleolin, another protein critical for rRNA processing, is involved in the biogenesis of microRNA 15a/16 (miR-15a/16), specifically at the primary to precursor stage of processing. Through overexpression and knockdown studies, we show that miR-15a/16 levels are directly correlated to nucleolin expression. Furthermore, we found that cellular localization is critical for the proper functioning of nucleolin in this pathway and that nucleolin directly interacts with DGCR8 and Drosha in the nucleus. Nucleolin can bind to the primary miRNA both directly and specifically. Finally, we show that in the absence of nucleolin, cell extracts are unable to process miR-15a/16 in vitro and that this can be rescued by the addition of nucleolin. Our findings offer a new protein component in the microRNA biogenesis pathway and lend insight into miRNA dysregulation in certain cancers.
微 RNA（miRNA）是一种内源性、短的、非编码 RNA，在生成功能成熟序列之前经历一个多步生物发生过程。微处理器复合物的核心组件，由 Drosha 和 DGCR8 组成，对于这个过程是必需和充分的，尽管已经发现了一些辅助蛋白，它们可以调节一部分 miRNA 的生物发生。奇怪的是，许多参与 miRNA 生物发生的蛋白质也需要参与核糖体 RNA 处理。在这里，我们表明核仁素，另一种对 rRNA 处理至关重要的蛋白质，参与 microRNA 15a/16（miR-15a/16）的生物发生，特别是在加工的初级到前体阶段。通过过表达和敲低研究，我们表明 miR-15a/16 的水平与核仁素表达直接相关。此外，我们发现细胞定位对于核仁素在该途径中的正常功能至关重要，并且核仁素在核内直接与 DGCR8 和 Drosha 相互作用。核仁素可以直接和特异性地结合到初级 miRNA 上。最后，我们表明在没有核仁素的情况下，细胞提取物无法在体外处理 miR-15a/16，并且可以通过添加核仁素来挽救。我们的发现为 miRNA 生物发生途径提供了一个新的蛋白质成分，并深入了解了某些癌症中 miRNA 失调的机制。
