Ben-Yishay Rakefet, Shav-Tal Yaron
The Mina & Everard Goodman Faculty of Life Sciences and the Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan, Israel.
Methods Mol Biol. 2019;2038:151-163. doi: 10.1007/978-1-4939-9674-2_10.
Export of mRNA transcripts from the cell nucleus is a complex and multistep process, regulated by various proteins and control mechanisms. Recent studies have demonstrated the rapid passage of mRNA-protein complexes (mRNPs) through the nuclear pore complex (NPC) as well as the ability to detect mRNPs stalled at the NPC during inhibition of the mRNA export process. In this chapter, we describe ways to block mRNA export and present an image analysis method to identify mRNPs stuck at the NPC during such blocks. Using the MS2 mRNA-tagging system to track single mRNPs in living cells we are able to examine their intracellular distribution and dynamics both in the nucleoplasm and at the nuclear periphery. We use this method to identify and count the number of static mRNPs anchored to the nuclear envelope under different conditions of mRNA export inhibition.
mRNA转录本从细胞核输出是一个复杂的多步骤过程，受多种蛋白质和调控机制的调节。最近的研究表明，mRNA-蛋白质复合物（mRNP）能快速通过核孔复合体（NPC），并且在mRNA输出过程受到抑制时，能够检测到停滞在NPC处 的mRNP。在本章中，我们描述了阻断mRNA输出的方法，并提出了一种图像分析方法，用于识别在这种阻断过程中滞留在NPC处的mRNP。利用MS2 mRNA标记系统追踪活细胞中的单个mRNP，我们能够研究它们在核质和核周的细胞内分布及动态变化。我们使用这种方法来识别和计数在不同mRNA输出抑制条件下锚定在核膜上的静态mRNP的数量。
