Molecular Mechanisms of mRNA Regulation and Decay

Division Of Basic Sciences - Nci

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Abstract

Overview and Objectives Over the last year, my lab has continued to investigated to investigate how host and pathogen factors co opt the CCR4/NOT deadenylase to control mRNA stability. The work focused on three mechanistic lines: (1) a Legionella pneumophila effector (PieF) that disabled host deadenylation; (2) Tristetraprolin (TTP/ZFP36) as a model RNA binding protein (RBP) that targeted CCR4/NOT to AU rich mRNAs; and (3) cooperative recognition and decay by the germline factors DND1 and NANOS. 1) Legionella PieF: Mechanism of Host Deadenylase Inhibition We reconstituted PieF complexes and confirmed a 1:1 stoichiometric interaction with the human deadenylase paralogs NOT7/8. PieF inhibited NOT7/8 catalytic activity in vitro but did not inhibit the complete CCR4/NOT complex or the isolated NOT7:NOT6 exonuclease module, indicating subcomplex specific antagonism. We determined high resolution cryo EM structures of PieF bound to NOT7:NOT1 and NOT8:NOT1. These <50 kDa single particle targets were technically challenging; success depended on access to state of the art cryo EM instrumentation in Frederick. The structures revealed that PieF simultaneously occluded the NOT7/8 active site and engaged a surface required for NOT7/8 association with NOT6/6L, thereby blocking assembly of the exonuclease module. Structure guided mutagenesis validated the binding mode and mechanism of inhibition. Upon PieF overexpression in HEK293 cells, direct RNA nanopore sequencing showed global poly(A) tail retention consistent with inhibition of deadenylation. Notably, PieF suppressed deadenylation more effectively than siRNA knockdown of NOT7/8 or NOT1, suggesting that PieF exploited paralog exchange or cotranslational association with subunits to disrupt formation of a deadenylation competent CCR4/NOT. The work uncovered a previously unrecognized pathogenesis strategy in which a bacterial effector directly targeted the host mRNA decay machinery. 2) TTP Targeting of CCR4/NOT and ARE Selective Deadenylation Using full length human TTP and fragments, we defined interactions with the NOT1 region within the NOT10:11 and NOT9 modules and identified an additional interaction with the NOT module. These contacts were mediated by TTP's N and C terminal intrinsically disordered regions (IDRs); the tandem zinc finger (TZF) RNA binding domain did not stably bind CCR4/NOT subunits under our assay conditions. In vitro deadenylation assays using the entire human CCR4/NOT complex demonstrated strong TTP dependent stimulation on AU rich (ARE) substrates relative to sequence matched mutants. A single point mutation in the TZF (C124R) abolished stimulation, confirming ARE recognition as essential. Fusions of the N or C terminal IDRs to the TZF also stimulated targeted deadenylation, indicating that multiple TTP regions contributed to CCR4/NOT recruitment and activation. An experienced postdoctoral fellow, Dr. Filip Pekovic, led this effort; he had previously reconstituted the Drosophila CCR4/NOT complex. These findings provided a unified view in which multivalent, phosphorylation sensitive IDR contacts (with NOT9/10/11 and NOT) cooperated with TZF mediated ARE binding to drive selective deadenylation. 3) DND1 NANOS Cooperation in Target Selection and Deadenylation In collaboration with Dr. Markus Hafner, PAR CLIP studies showed that DND1 bound AREs near 3' UTR ends, whereas NANOS2/3 alone lacked strong sequence specificity. When co expressed with DND1, NANOS2/3 gained pronounced specificity for the AUGAAUAA motif; tandem PAR CLIP indicated cooccupancy by the DND1:NANOS complex. The presence of this bipartite motif predicted transcript destabilization in human and mouse cells. Coexpression of DND1 with NANOS2/3 inhibited proliferation of HEK293 cells, consistent with repression of cell cycle mRNAs (e.g., CDK1). We produced full length human DND1 and NANOS2/3, mapped their interaction interfaces, and identified the minimal CCR4/NOT complement required for complex formation. In vitro assays demonstrated that the DND1:NANOS complex stimulated CCR4/NOT deadenylation more strongly than either factor alone on motif bearing RNAs. Guided by available structures (RNA bound DND1; Drosophila Nanos) and AlphaFold models, we initiated structure guided mutagenesis at protein protein interfaces and began thermodynamic and kinetic analyses of complex assembly. These studies established the experimental basis to quantify whether DND1 and NANOS cooperated kinetically and/or thermodynamically to create a high affinity, information rich cis element in 3'UTRs. The results supported a model in which interaction between unrelated RBPs increased sequence specificity and apparent affinity, enabling selective recruitment of CCR4/NOT to developmentally critical transcripts. Overall Impact Across pathogen, immune, and germline paradigms, we elucidated how small effectors and multivalent RBPs redirected or disabled the CCR4/NOT deadenylase. We defined a structural mechanism by which Legionella PieF blocks exonuclease module assembly and catalysis; we mapped and functionally validated multivalent TTP CCR4/NOT interactions that drive ARE selective decay; and we demonstrated cooperative DND1 NANOS recognition that enhances sequence specificity and stimulates deadenylation. Together, these accomplishments advanced a mechanistic framework for targeted deadenylation with direct relevance to infection biology, inflammation, cancer, and germline transcriptome remodeling.