CNOT4

Abstract: 4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis.

Abstract: Infection by most DNA viruses activates a cellular DNA damage response (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralize antiviral effects of DDR activation by targeting a number of proteins for rapid proteasome-mediated degradation. We have now identified a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1) (also known as Tab182), which is degraded during infection by adenovirus serotype 5 and adenovirus serotype 12. In both cases, degradation requires the action of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be serotype specific, as the protein remains relatively stable following infection with adenovirus serotypes 4, 7, 9, and 11. We have gone on to confirm that Tab182 is an integral component of the CNOT complex, which has transcriptional regulatory, deadenylation, and E3 ligase activities. The levels of at least 2 other members of the complex (CNOT3 and CNOT7) are also reduced during adenovirus infection, whereas the levels of CNOT4 and CNOT1 remain stable. The depletion of Tab182 with small interfering RNA (siRNA) enhances the expression of early region 1A proteins (E1As) to a limited extent during adenovirus infection, but the depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in the expression of adenovirus early proteins. In addition, the depletion of Tab182 and CNOT1 results in a limited increase in the viral DNA level during infection. We conclude that the cellular CNOT complex is a previously unidentified major target for adenoviruses during infection.IMPORTANCE Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have confirmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, the levels of other CNOT proteins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded, whereas CNOT4 and CNOT1 are not. The siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during infection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge, this is the first time that the CNOT complex has been identified as an adenoviral target.

Abstract: Understanding the molecular and functional interactions among macromolecular complexes, as well as their changes associated with time, cell type or disease state will be invaluable for human health, and will have direct implications, for example, in pharmaceutical research to identify and select potential targets, and design efficient and specific drugs. Structural studies of macromolecular complexes, however, suffer from some limitations, especially in the case of weak and transient complexes. New and complementary methodologies, such as docking, have therefore been developed. The current computational approaches, however, also suffer from limitations and new developments and improvements are needed. This thesis introduces a new docking approach in which the docking of two macromolecules is driven by biophysical and/or biochemical information and furthermore describes structural studies on the UbcH5B-CNOT4 complex involved in the ubiquitination pathway. HADDOCK is a new docking approach that is based on biophysical and/or biochemical information. This information, derived for example from NMR chemical shift perturbation or site-directed mutagenesis experiments, is converted into highly ambiguous intermolecular distance restraints that are directly used to drive the docking process. The docking protocol allows for side chains and backbone flexibility at the interface and the solutions are scored according to an intermolecular energy term. The method was successfully tested on three complexes. The solution NMR structure of UbcH5B, an E2 ubiquitin conjugating enzyme has been solved. NMR relaxation measurements are performed on UbcH5B. They show limited motions for the major part of the protein backbone. We compare the structure of UbcH5B with other E2 structures, and the global fold of all E2s is very similar. Some differences are, however, observed and correlate well with the dynamical properties of E2s. The position and orientation of the N-terminal a-helix as compared to the core of the protein differ in the various structures. This difference may be determinant in E3 ubiquitin ligase binding and recognition. Furthermore, a highly conserved asparagine residue was shown to be important for the ubiquitin transfer. In crystal structures, this asparagine points away from the active site cysteine. Structure of UbcH5B shows that in solution, this asparagine is in close proximity to the active site cysteine, in a conformation suitable for its catalytic role. HADDOCK is then used to generate a structural model of the UbcH5B-CNOT4 complex. CNOT4 is an E3 ubiquitin ligase that is part of the CCR4-NOT complex involved in transcription repression. The residues of the CNOT4 RING domain important for the interaction with UbcH5B were previously reported. Here, the residues of UbcH5B important for the binding to CNOT4 RING are identified from NMR chemical shift perturbation experiments. These data are used to generate a structural model of the UbcH5B-CNOT4 complex. Two sets of solutions are, however, obtained that can not be discriminated. Mutagenesis experiments are performed and identify charged residues of UbcH5B (Lys63) and of CNOT4 (Glu49) involved in an electrostatic interaction. Once this information is included in the docking, a unique set of solutions is obtained. The structural model of the UbcH5B-CNOT4 complex is compared with the X-ray structure of the homologous UbcH7-c-Cbl complex and significant differences at specific residues give structural insights into the mechanisms of the E2-E3 specificity.