Journal Article

Tagging enhances histochemical and biochemical detection of Ran Binding Protein 9 in vivo and reveals its interaction with Nucleolin

Stark, Aaron E., Breece, Chelssie C., Soliman, Shimaa H. A., La Perle, Krista M. D., Orlacchio, Arturo, Coppola, Vincenzo, Gardner, Miranda L., Freitas, Michael A., Tessari, Anna, Amari, Foued, Chen, Min, Palmieri, Dario, Harshman, Sean W., Martin, Jennifer A., Visone, Rosa

2020

Overview

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 38/1 / 38/23 / 38/77 / 42 / 42/41 / 631/1647/334/1874/345 / 631/80/86/2366 / 64/110 / 82/51 / 82/58 / Adaptor Proteins, Signal Transducing - genetics / Adaptor Proteins, Signal Transducing - metabolism / Animals / C-Terminus / CRISPR-Cas Systems / Cytoskeletal Proteins - genetics / Cytoskeletal Proteins - metabolism / Epitopes / Humanities and Social Sciences / Immunohistochemistry / Immunoprecipitation / Mass spectrometry / Mass spectroscopy / Mice / Mice, Knockout / multidisciplinary / Nuclear Proteins - genetics / Nuclear Proteins - metabolism / Nucleolin / Phenotypes / Phosphoproteins - metabolism / Protein Binding / Proteins / RNA, Messenger - metabolism / RNA-Binding Proteins - metabolism / Science / Science (multidisciplinary)