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Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay
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TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced

Xue-hai Liang1, Wen Shen2, Hong Sun2

  • 1Department of Core Antisense Research, ISIS Pharmaceuticals, Inc., Carlsbad, CA 92010, USA Lliang@isisph.com.

Nucleic Acids Research
|May 28, 2014
PubMed

Insights

The chaperonin T-complex 1 (TCP1) protein interacts with phosphorothioate antisense oligonucleotides (ASOs), enhancing their activity. TCP1 may also aid in ASO cellular uptake and nuclear import.

Area of Science:

  • Molecular Biology
  • Drug Discovery
  • Cell Biology

Background:

  • Phosphorothioate antisense oligonucleotides (PS-ASOs) are effective drugs for gene silencing via RNA degradation.
  • The interactions of PS-ASOs with cellular proteins beyond the RNase H pathway are not well understood.

Purpose of the Study:

  • To investigate the interactions between PS-ASOs and cellular proteins.
  • To elucidate the role of these interactions in PS-ASO activity and cellular localization.

Main Methods:

  • Cell transfection with PS-ASOs.
  • Co-localization studies using microscopy.
  • Analysis of PS-ASO localization upon depletion of specific proteins (e.g., RAN).

Main Results:

  • The chaperonin T-complex 1 (TCP1) proteins were found to interact with PS-ASOs and enhance their antisense activity.
  • TCP1-β subunit co-localized with PS-ASOs in nuclear structures called PS-bodies.
  • RAN depletion led to cytoplasmic PS-body-like structures and reduced nuclear PS-ASO concentration, suggesting RAN-mediated nuclear import.
  • Free uptake of PS-ASOs resulted in co-localization with TCP1 in endosomal/lysosomal compartments.

Conclusions:

  • The TCP1 complex binds to PS-ASOs, with TCP1-β being a key component of nuclear PS-bodies.
  • TCP1 may play a significant role in facilitating PS-ASO cellular uptake and their release from endocytic pathways.

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