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Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
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Detection of mRNAs Anchored to the Nuclear Envelope During Export Inhibition in Living Cells

Rakefet Ben-Yishay1, Yaron Shav-Tal2

  • 1The Mina & Everard Goodman Faculty of Life Sciences and the Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan, Israel.

Methods in Molecular Biology (Clifton, N.J.)
|August 14, 2019
PubMed

Insights

Researchers developed a method to visualize mRNA-protein complexes (mRNPs) stalled during nuclear export. This technique helps study the regulation of mRNA export by identifying mRNPs stuck at the nuclear pore complex (NPC).

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Nuclear mRNA export is a regulated, multistep process involving mRNA-protein complexes (mRNPs).
  • mRNPs rapidly transit the nuclear pore complex (NPC), but can stall during export inhibition.

Purpose of the Study:

  • To describe methods for blocking mRNA export and analyzing stalled mRNPs.
  • To utilize the MS2 mRNA-tagging system for tracking single mRNPs in living cells.

Main Methods:

  • Employing the MS2 mRNA-tagging system to visualize and track single mRNPs in real-time.
  • Developing an image analysis method to detect and quantify mRNPs stalled at the nuclear pore complex (NPC).
  • Inhibiting mRNA export to create conditions for mRNP stalling.

Main Results:

  • Successfully visualized the intracellular distribution and dynamics of single mRNPs.
  • Identified and counted static mRNPs anchored to the nuclear envelope under various export inhibition conditions.
  • Demonstrated the ability to detect mRNPs stalled at the NPC.

Conclusions:

  • The described methods allow for the identification and quantification of stalled mRNPs at the NPC.
  • This approach provides insights into the dynamics and regulation of mRNA export.
  • Facilitates the study of mRNA transport mechanisms and potential disruptions.
Keywords:
Gene expressionImage processing and analysisLive cell imagingNuclear pore complex (NPC)Single particle trackingmRNA exportmRNP

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