Structure and function of molecular machines involved in deadenylation-dependent 5'-3' mRNA degradation.

In eukaryotic cells, the synthesis, processing, and degradation of mRNA are important processes required for the accurate execution of gene expression programmes. Fully processed cytoplasmic mRNA is characterised by the presence of a 5'cap structure and 3'poly(A) tail. These elements promote translation and prevent non-specific degradation. Degradation via the deadenylation-dependent 5'-3' degradation pathway can be induced by trans-acting factors binding the mRNA, such as RNA-binding proteins recognising sequence elements and the miRNA-induced repression complex. These factors recruit the core mRNA degradation machinery that carries out the following steps: i) shortening of the poly(A) tail by the Ccr4-Not and Pan2-Pan3 poly (A)-specific nucleases (deadenylases); ii) removal of the 5'cap structure by the Dcp1-Dcp2 decapping complex that is recruited by the Lsm1-7-Pat1 complex; and iii) degradation of the mRNA body by the 5'-3' exoribonuclease Xrn1. In this review, the biochemical function of the nucleases and accessory proteins involved in deadenylation-dependent mRNA degradation will be reviewed with a particular focus on structural aspects of the proteins and enzymes involved.

Regulation of eukaryotic mRNA deadenylation and degradation by the Ccr4-Not complex.

Accurate and precise regulation of gene expression programmes in eukaryotes involves the coordinated control of transcription, mRNA stability and translation. In recent years, significant progress has been made about the role of sequence elements in the 3' untranslated region for the regulation of mRNA degradation, and a model has emerged in which recruitment of the Ccr4-Not complex is the critical step in the regulation of mRNA decay. Recruitment of the Ccr4-Not complex to a target mRNA results in deadenylation mediated by the Caf1 and Ccr4 catalytic subunits of the complex. Following deadenylation, the 5' cap structure is removed, and the mRNA subjected to 5'-3' degradation. Here, the role of the human Ccr4-Not complex in cytoplasmic deadenylation of mRNA is reviewed, with a particular focus on mechanisms of its recruitment to mRNA by sequence motifs in the 3' untranslated region, codon usage, as well as general mechanisms involving the poly(A) tail.

Structural Model of the Human BTG2-PABPC1 Complex by Combining Mutagenesis, NMR Chemical Shift Perturbation Data and Molecular Docking.

Degradation of cytoplasmic mRNA in eukaryotes involves the shortening and removal of the mRNA poly(A) tail by poly(A)-selective ribonuclease (deadenylase) enzymes. In human cells, BTG2 can stimulate deadenylation of poly(A) bound by cytoplasmic poly(A)-binding protein PABPC1. This involves the concurrent binding by BTG2 of PABPC1 and the Caf1/CNOT7 nuclease subunit of the Ccr4-Not deadenylase complex. To understand in molecular detail how PABPC1 and BTG2 interact, we set out to identify amino acid residues of PABPC1 and BTG2 contributing to the interaction. To this end, we first used algorithms to predict PABPC1 interaction surfaces. Comparison of the predicted interaction surface with known residues involved in the binding to poly(A) resulted in the identification of a putative interaction surface for BTG2. Subsequently, we used pulldown assays to confirm the requirement of PABPC1 residues for the interaction with BTG2. Analysis of RNA-binding by PABPC1 variants indicated that PABPC1 residues required for interaction with BTG2 do not interfere with poly(A) binding. After further defining residues of BTG2 that are required for the interaction with PABPC1, we used information from published NMR chemical shift perturbation experiments to guide docking and generate a structural model of the BTG2-PABPC1 complex. A quaternary poly(A)-PABPC1-BTG2-Caf1/CNOT7 model showed that the 3' end of poly(A) RNA is directed towards the catalytic centre of Caf1/CNOT7, thereby providing a rationale for enhanced deadenylation by Caf1/CNOT7 in the presence of BTG2 and PABPC1.

Structure of the human Ccr4-Not nuclease module using X-ray crystallography and electron paramagnetic resonance spectroscopy distance measurements.

Regulated degradation of mature, cytoplasmic mRNA is a key step in eukaryotic gene regulation. This process is typically initiated by the recruitment of deadenylase enzymes by cis-acting elements in the 3' untranslated region resulting in the shortening and removal of the 3' poly(A) tail of the target mRNA. The Ccr4-Not complex, a major eukaryotic deadenylase, contains two exoribonuclease subunits with selectivity toward poly(A): Caf1 and Ccr4. The Caf1 deadenylase subunit binds the MIF4G domain of the large subunit CNOT1 (Not1) that is the scaffold of the complex. The Ccr4 nuclease is connected to the complex via its leucine-rich repeat (LRR) domain, which binds Caf1, whereas the catalytic activity of Ccr4 is provided by its EEP domain. While the relative positions of the MIF4G domain of CNOT1, the Caf1 subunit, and the LRR domain of Ccr4 are clearly defined in current models, the position of the EEP nuclease domain of Ccr4 is ambiguous. Here, we use X-ray crystallography, the AlphaFold resource of predicted protein structures, and pulse electron paramagnetic resonance spectroscopy to determine and validate the position of the EEP nuclease domain of Ccr4 resulting in an improved model of the human Ccr4-Not nuclease module.

1-Hydroxy-xanthine derivatives inhibit the human Caf1 nuclease and Caf1-containing nuclease complexes via Mg2+-dependent binding.

In eukaryotic cells, cytoplasmic mRNA is characterised by a 3' poly(A) tail. The shortening and removal of poly(A) tails (deadenylation) by the Ccr4-Not nuclease complex leads to reduced translational efficiency and RNA degradation. Using recombinant human Caf1 (CNOT7) enzyme as a screening tool, we recently described the discovery and synthesis of a series of substituted 1-hydroxy-3,7-dihydro-1H-purine-2,6-diones (1-hydroxy-xanthines) as inhibitors of the Caf1 catalytic subunit of the Ccr4-Not complex. Here, we used a chemiluminescence-based AMP detection assay to show that active 1-hydroxy-xanthines inhibit both isolated Caf1 enzyme and human Caf1-containing complexes that also contain the second nuclease subunit Ccr4 (CNOT6L) to a similar extent, indicating that the active site of the Caf1 nuclease subunit does not undergo substantial conformational change when bound to other Ccr4-Not subunits. Using differential scanning fluorimetry, we also show that binding of active 1-hydroxy-xanthines requires the presence of Mg2+ ions, which are present in the active site of Caf1.

MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo.

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.

The central region of CNOT1 and CNOT9 stimulates deadenylation by the Ccr4-Not nuclease module.

Regulated degradation of cytoplasmic mRNA is important for the accurate execution of gene expression programmes in eukaryotic cells. A key step in this process is the shortening and removal of the mRNA poly(A) tail, which can be achieved by the recruitment of the multi-subunit Ccr4-Not nuclease complex via sequence-specific RNA-binding proteins or the microRNA machinery. The Ccr4-Not complex contains several modules that are attached to its large subunit CNOT1. Modules include the nuclease module, which associates with the MIF4G domain of CNOT1 and contains the catalytic subunits Caf1 and Ccr4, as well as the module containing the non-catalytic CNOT9 subunit, which binds to the DUF3819 domain of CNOT1. To understand the contributions of the individual modules to the activity of the complex, we have started to reconstitute sub-complexes of the human Ccr4-Not complex containing one or several functional modules. Here, we report the reconstitution of a pentameric complex including a BTG2-Caf1-Ccr4 nuclease module, CNOT9 and the central region of CNOT1 encompassing the MIF4G and DUF3819 domains. By comparing the biochemical activities of the pentameric complex and the nuclease module, we conclude that the CNOT1-CNOT9 components stimulate deadenylation by the nuclease module. In addition, we show that a pentameric complex containing the melanoma-associated CNOT9 P131L variant is able to support deadenylation similar to a complex containing the wild-type CNOT9 protein.