Mechanistic insights into the regulation of DEAD-box helicases Sub2 and UAP56 by Tho1-family proteins

Eukaryotic mRNA is transcribed, processed, and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus before being exported to the cytoplasm for translation. The highly conserved TREX complex plays a central role in coordinating these events. In baker’s yeast Saccharomyces cerevisiae, defects in the largest TREX subunit, the THO complex, result in hyperrecombination, impaired mRNA export, and growth defects. Notably, these phenotypes can be suppressed by overexpression of the DEAD-box RNA helicase and TREX component Sub2 or its interaction partner Tho1. Despite increasing interest in TREX and its associated factors in the past years, the molecular mechanisms by which Tho1, in particular, fulfils its cellular functions remain unclear. Recent structural studies revealed a 2:1 complex formed by two protomers of the human Sub2 ortholog DDX39B and the C-terminal domain (CTD) of Tho1. However, the functional and physiological relevance of their interaction is not yet understood. This study provides a comprehensive biochemical characterisation of Tho1 and its A. thaliana ortholog MOS11, elucidating the molecular mechanism by which they stimulate unwinding by Sub2 or its A. thaliana ortholog UAP56. Building on the prior identification of a nucleic acid annealing activity of Tho1, the functional contributions of individual Tho1 domains were assessed using quantitative fluorescence-based real-time binding and annealing assays. Importantly, different Tho1 truncation mutants were tested for their ability to suppress a temperature-sensitive growth defect in cells lacking the THO component Hpr1, revealing that the Tho1-CTD alone is sufficient for phenotype suppression. Strikingly, while the Tho1-CTD lacks intrinsic nucleic acid binding and annealing activities, it interacts with and stimulates Sub2’s helicase activity. To dissect the mechanism underlying this helicase stimulation, fluorescence-based real-time unwinding assays were combined with complex modelling and mutational analyses. The Tho1-CTD contains two conserved α-helical motifs, each capable of interacting with one helicase protomer, and both motifs were shown to be crucial for helicase stimulation. Evolutionary conservation of this mechanism was confirmed by demonstrating that the Tho1-CTD stimulates UAP56. Furthermore, crosslinking approaches showed that formation of a 2:1 complex is critical for helicase stimulation. Together, these findings reveal that Tho1 acts as a molecular scaffold promoting helicase oligomerisation and thereby activation. The physiological importance of this mechanism is underscored by the requirement for two intact helical motifs within the Tho1-CTD to suppress the growth defect in ∆hpr1 cells. Further investigation of MOS11, which contains five helical motifs connected by flexible linker regions, provided additional insight into how both the number of motifs and their spatial arrangement influence scaffold function. These structural features define its effective reach, which in turn determines the range of substrates on which helicase stimulation can be observed. Collectively, this work combines in vitro biochemical assays, in silico modelling, and in vivo studies to provide mechanistic insights into the interaction of Tho1-family proteins and their role as cofactors that facilitate DEAD-box helicase oligomerisation.

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Mechanistic insights into the regulation of DEAD-box helicases Sub2 and UAP56 by Tho1-family proteins

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