A Concentration-Dependent Liquid Phase Separation Can Cause Toxicity upon Increased Protein Expression

Abstract

SummaryMultiple human diseases are associated with a liquid-to-solid phase transition resulting in the formation of amyloid fibers or protein aggregates. Here, we present an alternative mechanism for cellular toxicity based on a concentration-dependent liquid-liquid demixing. Analyzing proteins that are toxic when their concentration is increased in yeast reveals that they share physicochemical properties with proteins that participate in physiological liquid-liquid demixing in the cell. Increasing the concentration of one of these proteins indeed results in the formation of cytoplasmic foci with liquid properties. Demixing occurs at the onset of toxicity and titrates proteins and mRNAs from the cytoplasm. Focus formation is reversible, and resumption of growth occurs as the foci dissolve as protein concentration falls. Preventing demixing abolishes the dosage sensitivity of the protein. We propose that triggering inappropriate liquid phase separation may be an important cause of dosage sensitivity and a determinant of human disease.

The RNA binding protein Mip6, a novel cellular partner of Mex67 export factor with implications in mRNA export

“…It might be proposed then, that the RNA binding Mip6 is also playing that role, and while it binds Mex67 through its RRM4, the other RRMs are still binding RNA as we showed and thus would be holding RNA and further shuttling with Mex67 to the nucleus especially as Mip6 doesn't have the capacity to shuttle on its own. This hypothesis is also supported by the fact that in vivo Mip6 localizes homogeneously both in nucleus and in cytoplasm at normal growth conditions (Bolognesi et al, 2016), however when Mex67 was inactivated in vivo, most Mip6-GFP was retained in the nucleus. Additionally, a non-published work from in Susana Rodriguez Navarro lab have also provided evidence for Mip6 co-purifying with proteins involved in mRNA export and known to interact with Mex67 as it exports the nucleus like Sac3 (a part of the TREX-2 complex on the nuclear face of the NPC), and Dbp5 (localizes at the cytoplasmic face of the NPC) using TAP purification.…”

“…One study that looked at the cellular toxicity caused by a concentration-dependent liquid phase separation upon increased protein expression, suggested that the strong over-expression of the dosage-sensitive Mip6 protein indeed changes its localization to cytoplasmic foci impairing cell growth, reducing the global translation rates, and causing toxicity (Bolognesi et al, 2016). The study also found that these cytoplasmic foci contained RNA and demonstrated that at least two of the RRM domains of Mip6 are required for the cytoplasmic focus formation.…”

“…However though, a recent study on protein concentration dependent cellular toxicity, suggested that over expression of the yeast dosage sensitive Mip6 protein causes the protein to localize, reversibly, to cytoplasmic RNP granules causing a reduction in the global translation rates and thus triggering cellular toxicity and growth impairment when strongly over expressed (Bolognesi et al, 2016).…”

“…In the only two papers on the subject, The "predicted" RRM domains of Mip6 or both Mip6 and Pes4, are said to be critical for their function: formation of cytoplasmic foci containing RNA when Mip6 is over-expressed (Bolognesi et al, 2016), and the role of both Mip6 and Pes4 in mRNA protection and localization in stress conditions (Jin et al, 2017). However though, no one had actually demonstrated the ability of these proteins to bind RNA in vitro or in vivo or looked closer at the number and diversity of RRMs they contained.…”

“…In the very few literature references on Mip6 or Pes4, the proteins were usually referred to as putative RNA binding proteins containing three RRMs (Bolognesi et al, 2016;Jin, Zhang, Sternglanz, & Neiman, 2017;Segref et al, 1997). In the only two papers on the subject, The "predicted" RRM domains of Mip6 or both Mip6 and Pes4, are said to be critical for their function: formation of cytoplasmic foci containing RNA when Mip6 is over-expressed (Bolognesi et al, 2016), and the role of both Mip6 and Pes4 in mRNA protection and localization in stress conditions (Jin et al, 2017).…”