Fig. 1: Legionella effector PieF binds NOT7 and NOT8 deadenylase enzymes.
From: Intracellular pathogen effector reprograms host gene expression by inhibiting mRNA decay
a Domain organization and schematic representation of the catalytic module of the human CCR4-NOT complex. b Coomassie-stained 15% polyacrylamide gel of the in vitro streptavidin pull-down assay with recombinant N-terminally StrepII-tagged PieF upon incubation with NOT9 module and His6-SUMO-tagged NOT7 and NOT8 deadenylases. * denotes the presence of an N-terminal His6-SUMO-tag. # denotes an N-terminal His6-MBP-tag. Experiment was independently repeated multiple times (n > 3). c Coomassie-stained 15% polyacrylamide gel of the in vitro streptavidin pull-down assay with recombinant N-terminally StrepII-tagged PieF upon incubation with His6-SUMO-tagged NOT7*, NOT6:NOT7*** and NOT6L:NOT7** heterodimers. * denotes presence of an N-terminal His6-SUMO-tag, ** denotes an N-terminal His8-tag. *** denotes untagged NOT7. Experiment was independently repeated multiple times (n > 3). d, e In vitro deadenylation assays with 50 nM of UCUACAU-A20 RNA substrate, His6-SUMO-NOT7 (500 nM; d) and NOT6:NOT7 heterodimer (250 nM; e) without (left panels) and with (right panels) the equimolar to corresponding deadenylase amount of N-terminally His6-tagged PieF. M indicates the tail length RNA marker, and C represents the control sample without the deadenylase. Poly (A) tail length changes were quantified by plotting the most abundant tail length at each time point. Linear regression was used to determine the apparent deadenylation rate (As/min); values are presented as mean ± SE (n = 3). The molecular size markers of right panels are identical to the ones on the left. f Representative isothermal titration calorimetry (ITC) thermograms of the interaction between His6-tagged PieF and His6-SUMO-NOT7. The upper panel shows raw data in (µcal s−1), and the lower panel represents the integration of heat changes associated with each injection (kcal mol−1 of injectant). Data were fitted using a one-site binding model. The parameters of the runs are summarized in Supplementary Table 1. The schematics in (a, d, e) were drawn using Adobe Illustrator 2025. Source data are provided as a Source Data file.
