Discovery of Substituted 5-(2-Hydroxybenzoyl)-2-Pyridone Analogues as Inhibitors of the Human Caf1/CNOT7 Ribonuclease.

The Caf1/CNOT7 nuclease is a catalytic component of the Ccr4-Not deadenylase complex, which is a key regulator of post-transcriptional gene regulation. In addition to providing catalytic activity, Caf1/CNOT7 and its paralogue Caf1/CNOT8 also contribute a structural function by mediating interactions between the large, non-catalytic subunit CNOT1, which forms the backbone of the Ccr4-Not complex and the second nuclease subunit Ccr4 (CNOT6/CNOT6L). To facilitate investigations into the role of Caf1/CNOT7 in gene regulation, we aimed to discover and develop non-nucleoside inhibitors of the enzyme.

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.

CCR4-NOT differentially controls host versus virus poly(a)-tail length and regulates HCMV infection.

Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host translatome without producing an mRNA decay enzyme. By performing a targeted loss-of-function screen in primary human fibroblasts, we here identify the host CCR4-NOT deadenylase complex members CNOT1 and CNOT3 as unexpected pro-viral host factors that selectively regulate HCMV reproduction. We find that the scaffold subunit CNOT1 is specifically required for late viral gene expression and genome-wide host responses in CCR4-NOT-disrupted cells.

Quantitative Biochemical Analysis of Deadenylase Enzymes Using Fluorescence and Chemiluminescence-Based Assays.

Deadenylase enzymes play a key role in mRNA degradation and RNA processing. In this chapter, we describe two activity assays for the quantitative biochemical analysis of deadenylase enzymes, which can easily be adapted for other nuclease enzymes. The assays use distinct principles of detection, which are based on differential annealing of a probe complementary to the substrate RNA or detection of adenosine monophosphate (AMP).

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.

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.

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.

Design of artificial metalloenzymes for the reduction of nicotinamide cofactors.

Artificial metalloenzymes result from the insertion of a catalytically active metal complex into a biological scaffold, generally a protein devoid of other catalytic functionalities. As such, their design requires efforts to engineer substrate binding, in addition to accommodating the artificial catalyst. Here we constructed and characterised artificial metalloenzymes using alcohol dehydrogenase as starting point, an enzyme which has both a cofactor and a substrate binding pocket.

Frequent loss of BTG1 activity and impaired interactions with the Caf1 subunit of the Ccr4-Not deadenylase in non-Hodgkin lymphoma.

Mutations in the highly similar genes B-cell translocation gene 1 () and are identified in approximately 10-15% of non-Hodgkin lymphoma cases, which may suggest a direct involvement of and in malignant transformation. However, it is unclear whether or how disease-associated mutations impair the function of these genes. Therefore, we selected 16 BTG1 variants based on analysis.

1-Hydroxy-xanthine derivatives inhibit the human Caf1 nuclease and Caf1-containing nuclease complexes via Mg-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-1-purine-2,6-diones (1-hydroxy-xanthines) as inhibitors of the Caf1 catalytic subunit of the Ccr4-Not complex.

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.