Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724, USA.
Nat Cell Biol. 2011 Jan;13(1):95-101. doi: 10.1038/ncb2140. Epub 2010 Dec 19.
The cell nucleus is a highly compartmentalized organelle harbouring a variety of dynamic membraneless nuclear bodies. How these subnuclear domains are established and maintained is not well understood. Here, we investigate the molecular mechanism of how one nuclear body, the paraspeckle, is assembled and organized. Paraspeckles are discrete ribonucleoprotein bodies found in mammalian cells and implicated in nuclear retention of hyperedited mRNAs. We developed a live-cell imaging system that allows for the inducible transcription of Men ɛ/β (also known as Neat1; ref. 12) noncoding RNAs (ncRNAs) and the direct visualization of the recruitment of paraspeckle proteins. Using this system, we demonstrate that Men ɛ/β ncRNAs are essential to initiate the de novo assembly of paraspeckles. These newly formed structures effectively harbour nuclear-retained mRNAs confirming that they are bona fide functional paraspeckles. By three independent approaches, we show that it is the act of Men ɛ/β transcription, but not ncRNAs alone, that regulates paraspeckle maintenance. Finally, fluorescence recovery after photobleaching (FRAP) analyses supported a critical structural role for Men ɛ/β ncRNAs in paraspeckle organization. This study establishes a model in which Men ɛ/β ncRNAs serve as a platform to recruit proteins to assemble paraspeckles.
细胞核是一个高度分隔的细胞器，其中包含各种动态的无膜核体。这些亚核区是如何建立和维持的，目前还不是很清楚。在这里，我们研究了一个核体——核斑（paraspeckle）组装和组织的分子机制。核斑是哺乳动物细胞中发现的离散核糖核蛋白体，与超编辑 mRNA 的核保留有关。我们开发了一种活细胞成像系统，可以诱导转录 Men ɛ/β（也称为 Neat1；参考文献 12）非编码 RNA（ncRNA），并直接可视化核斑蛋白的募集。使用该系统，我们证明 Men ɛ/β ncRNA 对于从头组装核斑是必不可少的。这些新形成的结构有效地包含了核保留的 mRNA，证实了它们是真正的功能性核斑。通过三种独立的方法，我们表明是 Men ɛ/β 转录的作用，而不仅仅是 ncRNA 本身，调节核斑的维持。最后，荧光恢复后光漂白（FRAP）分析支持 Men ɛ/β ncRNA 在核斑组织中的关键结构作用。这项研究建立了一个模型，即 Men ɛ/β ncRNA 作为一个平台，招募蛋白质组装核斑。
