BAKHTIARI, Ashkan Karimi
(2025)
Human CNOT3 in Cell Proliferation and Disease: Insights into the Function of the Ccr4-Not Complex.
PhD thesis, University of Nottingham.
Abstract
The Ccr4-Not complex, a conserved multi-subunit regulator of mRNA metabolism, transcription, and translation, plays a pivotal role in maintaining cellular homeostasis. The work described in this thesis investigated the role of human CNOT3, a core subunit of the Ccr4-Not complex. The protein contains two conserved domains linked by an intrinsically disordered region. The N-terminal domain can directly interact with the ribosome, while the C-terminal Not-Box region is required for incorporation into the Ccr4-Not complex, which involves interactions with the C-terminal domain of the CNOT1 subunit and the Not-Box region of CNOT2. To examine the structural and functional significance of CNOT3, stable HEK293 cell lines with inducible expression of siRNA-resistant CNOT3 variants were generated. These cell lines allowed dissection of CNOT3’s role in cell proliferation by siRNA-mediated knockdown of endogenous CNOT3 combined with overexpression of siRNA-resistant CNOT3 variants. Western blot analyses confirmed that the CNOT3 variants studied were expressed at comparable levels, validating the functionality of the experimental system.
The use of AlamarBlue and crystal violet cell viability assays revealed that deletion of the N-terminal and C-terminal domains of CNOT3 failed to rescue cell proliferation following siRNA knockdown of endogenous CNOT3, highlighting their essential roles in efficient cell proliferation in the HEK293 cell model. Furthermore, co-immunoprecipitation experiments revealed that the C-terminal domain is essential for the incorporation of CNOT3 into the Ccr4-Not complex, whereas the N-terminal domain is not required for this interaction. These findings demonstrate the importance of the structural integrity of CNOT3 in promoting cellular growth and maintaining its incorporation into the complex.
Mutations in CNOT3, particularly within its N-terminal domain, have been associated with T-cell acute lymphoblastic leukaemia (T-ALL) and neurodevelopmental disorders (NDDs). This research integrates computational tools, such as SIFT, PolyPhen-2, PROVEAN, and AlphaMissense, with experimental validation to evaluate the impact of missense mutations. While these mutations preserved the fundamental interactions within the Ccr4-Not complex, they appear to modulate its function through subtle mechanisms. Specifically, given the established role of the N-terminal domain in ribosome binding, these mutations may influence both regulatory pathways and translational efficiency.
In addition, mutations in CNOT2 were investigated, demonstrating that truncating mutations result in haploinsufficiency by disrupting its incorporation into the Ccr4-Not complex, as shown through co-immunoprecipitation experiments using plasmid-based expression systems. In contrast, the missense mutation does not affect core interactions but may have subtler functional consequences that require further study. These findings contribute to understanding how mutations in CNOT3 and CNOT2 influence the structural and functional integrity of the Ccr4-Not complex. By providing a foundation for future investigations into their broader biological and pathological significance, this work emphasises the need for detailed functional studies to elucidate the precise mechanisms underlying these mutations’ contributions to disease.
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