Detection of mRNAs Anchored to the Nuclear Envelope During Export Inhibition in Living Cells
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.
Key wordsmRNP mRNA export Nuclear pore complex (NPC) Gene expression Live cell imaging Single particle tracking Image processing and analysis
Y.S.T. is supported by the Israel Science Foundation and German–Israeli Foundation.
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