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Related Concept Videos

Regulation of Nuclear Protein Sorting01:45

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Nuclear protein sorting regulates nucleus composition and gene expression, crucial for determining the fate of a eukaryotic cell. Hence, the entry and exit of molecules across the nuclear envelope is a tightly controlled process. Nuclear protein sorting can be inhibited by one of the following ways: 1) masking cargo signal sequences, 2) modifying the nuclear receptor's affinity for cargo, 3) controlling the nuclear pore size, 4) retaining the cargo during its transit to the cytosol or the...
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Proteins targeted to the nucleus carry short stretches of amino acid sequences called the nuclear localization signal or NLS. Classical nuclear localization signals are of two types: monopartite and bipartite NLS. Monopartite classical NLS (cNLS) consists of a single cluster of 4-8 amino acids. Bipartite cNLS consists of two clusters of  2-3 amino acids and a 9-12 residue long proline-rich linker bridging the two clusters. Signal clusters are rich in positively charged amino acids such as...
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Nuclear protein sorting is the selective trafficking of histones, polymerases, gene regulatory proteins into the nucleus and exporting RNAs and ribosomes to the cytosol. It is a tightly controlled process that regulates gene expression within a cell.
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Analysis of Nucleoporin Function Using Inducible Degron Techniques.

Vasilisa Aksenova1, Alexei Arnaoutov2, Mary Dasso2

  • 1Division of Molecular and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA. vasilisa.aksenova@nih.gov.

Methods in Molecular Biology (Clifton, N.J.)
|April 12, 2022
PubMed
Summary
This summary is machine-generated.

The auxin-inducible degron (AID) system enables rapid protein depletion for studying complex cellular machinery like the nuclear pore complex (NPC). This protocol details CRISPR/Cas9 gene editing for AID tagging nucleoporins, facilitating research into NPC function and assembly.

Keywords:
Auxin-Inducible DegradationCRISPR/Cas9Nuclear Pore Complex

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • The auxin-inducible degron (AID) system offers precise control over protein stability.
  • Studying large protein complexes like the nuclear pore complex (NPC) presents challenges for traditional methods.
  • Acute protein depletion is crucial for dissecting roles in complex assembly and function.

Purpose of the Study:

  • To describe a protocol for CRISPR/Cas9-mediated gene targeting using the AID system.
  • To provide a detailed example for tagging the NUP153 gene within the NPC.
  • To offer guidance on screening and integrating the TIR1 component for AID-dependent degradation.

Main Methods:

  • CRISPR/Cas9 gene editing for AID tagging of target genes.
  • Step-by-step protocol for NUP153 gene targeting.
  • Recommendations for screening and TIR1 integration.

Main Results:

  • Successful demonstration of CRISPR/Cas9 for AID tagging of nucleoporins.
  • Established a method for acute depletion of individual NPC components.
  • Provided a framework for analyzing NUP-AID tagged cell lines.

Conclusions:

  • The NUP-AID system provides a powerful tool for studying nucleoporin function.
  • This protocol facilitates research into nuclear pore complex assembly and dynamics.
  • The described methods enable precise investigation of protein roles in cellular processes.