Fig. 4: Validation of the PieF inhibitory interface in deadenylation assays and in cells.
From: Intracellular pathogen effector reprograms host gene expression by inhibiting mRNA decay
a Schematic representation of the mutated residues in PieF used in this study. b In vitro deadenylation assays with 50 nM of UCUACAU-A20 RNA substrate, 500 nM of His6-SUMO-NOT7 and 500 nM of His6-tagged PieFK124A, PieFK124R, PieF 5 M, PieF 2 M, and PieF 3 M. The molecular size markers on all gels are identical to those shown on the leftmost panel. Experiment was independently repeated multiple times (n = 3). Source data are provided as a Source Data file. c Violin plots showing the poly(A) tail distribution in HEK293T cells expressing transfected GFP control (green), GFP-NOT7D40A (blue), GFP-PieF (dark red), and GFP-PieF 5M (orange) based on mRNA poly(A) length estimates by nanopore sequencing. Modal poly(A) tail length values are next to the corresponding violin plot. Box plots show median value (solid bold line) and 1st and 3rd quartiles, and whiskers represent 1.5 × IQR (interquartile range). n is the number of basecalled reads in each sample. d Cumulative cell counts at day 2, day 4, and day 6 after transfection and overexpression of a control GFP construct (green) compared to GFP-NOT7D40A (blue), GFP-PieF WT (dark red) or GFP-PieF 5 M (orange) constructs. Values are presented as mean ± standard deviation (n = 3). Source data are provided as a Source Data file. e The Legionella pneumophila effector PieF or NOT6 bind to NOT7 within the CCR4-NOT complex in a mutually exclusive manner. PieF binding impairs the assembly and deadenylation activity of the CCR4-NOT complex, leading to longer poly(A) tails, increased mRNA levels, and defects in cell cycle progression. The schematics were drawn using Adobe Illustrator 2025.
