Intracellular pathogen effector reprograms host gene expression by inhibiting mRNA decay

Abstract

Legionella pneumophila, an intracellular bacterial pathogen, injects effector proteins into host cells to manipulate cellular processes and promote its survival and proliferation. Here, we reveal a unique mechanism by which the Legionella effector PieF perturbs host mRNA decay by targeting the human CCR4-NOT deadenylase complex. High-resolution cryo-electron microscopy structures and biochemical analyses reveal that PieF binds with nanomolar affinity to the NOT7 and NOT8 catalytic subunits of CCR4-NOT, obstructing RNA access and displacing a catalytic Mg²⁺ ion from the active site. Additionally, PieF prevents NOT7/8 from associating with their partner deadenylases NOT6/6L, inhibiting the assembly of a functional deadenylase complex. Consequently, PieF robustly blocks mRNA poly(A) tail shortening and degradation with striking potency and selectivity for NOT7/8. This inhibition of deadenylation by PieF impedes cell cycle progression in human cells, revealing a novel bacterial strategy to modulate host gene expression.

Fig. 1. Legionella effector PieF binds NOT7 and NOT8 deadenylase enzymes.

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.

Fig. 2. Reconstitution and cryo-EM structures of the NOT1:NOT7/8:PieF ternary complexes.

a Analytical size exclusion chromatography profiles (left panel) and a 15% Coomassie-stained gel (right panel) of the purified components and intermediates of the NOT1:NOT7:PieF complex. The tags used to purify the individual components were preserved during the reconstitution of the complex. Experiment was independently repeated multiple times (n > 3). Source data are provided as a Source Data file. b Mass photometry analysis of the NOT1:NOT7:PieF complex. The tags used for purification were preserved during the reconstitution of the complex. c Cryo-EM reconstruction of the NOT1:NOT7:PieF complex at 2.8 Å resolution. d Cryo-EM reconstruction of the NOT1:NOT8:PieF complex at 3.5 Å resolution. e Superimposed structures of the NOT1:NOT7/8:PieF complexes in cartoon representation. f Secondary structure annotation of PieF structure. The schematics in (a, b) were drawn using Adobe Illustrator 2025.

Fig. 3. Structural details of the NOT7:PieF interfaces.

a Cartoon representation of the NOT7 (blue) active site and part of the helix α3 of PieF (red). Residues K206–E211 were omitted for clarity. The residues involved in the interaction are represented as sticks and the Mg²⁺ atom is shown as a green sphere. Dashed lines indicate metal coordination (NOT7 and Mg²⁺) or hydrogen bonding (NOT7 and PieF). D40^NOT7 and D161^NOT7 are additionally involved in salt bridge interactions with K124^PieF. Interaction of K124^PieF with D40^NOT7 and D161^NOT7 displaces a catalytic Mg²⁺ from the active site, and the NOT7 crystal structure (PDB: 4gmj; transparent blue) is superimposed to illustrate this. b Close-up view of (a). c NOT7:PieF, as represented in (a), superimposed on the crystal structure of the NOT6:NOT7 heterodimer (PDB: 7ax1). The leucine-rich repeat region of NOT6 binds to the same site of NOT7 as PieF. The N-terminal region of NOT6 (solid yellow) extends the binding interface. d Close-up view of (c).

Fig. 4. Validation of the PieF inhibitory interface in deadenylation assays and in cells.

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 PieF^K124A, PieF^K124R, 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-NOT7^D40A (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-NOT7^D40A (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.