Development of the microtubule bench
To obtain structural data on protein or protein:RNA interactions in the cell, we have developed a new technology based on the expertise of the laboratory in microtubule dynamics (Patent, WO 2016012451 A1, 2016). The principle is simple: a protein that serves as bait is brought onto microtubules in mammalian cells thought its fusion to a microtubule-binding domain. The presence of a protein prey on microtubules then reveals an interaction between bait and prey. This technology present major advantages compared to existing methods:
i) detection is carried out in living cells without the need for antibodies, unlike proximity ligation assay,
ii) the interactions can be measured quantitatively and according to the level of expression of the bait and the prey and compared with each other (impossible with the FRET because the signal depends on the couple considered and the orientation and distance between the donor and acceptor).
Probing and measuring liquid/liquid phase separation
Of course, there are disadvantages associated with the fusion of the bait to a microtubule binding domain. However, we have shown that the fusion of RNA-binding proteins to tau, a microtubule-binding protein, and the vicinity of microtubules do not prevent their interactions with RNA in cells. In addition, the consequences of point mutations on interactions between RNA-binding proteins can be probed which provides a mean to decipher the structural basis of protein:RNA interactions at the cellular level. Combining cellular data obtained via the microtubule bench with in vitro data provided by NMR spectroscopy is thus promising.
Very recently, we have adapted this technology to analyze and visualize phase separations orchestrated by RNA binding proteins.