[PhD Thesis Presentation] ‐ Ms. Dina Mostafa "Functional analysis of CCR4-NOT complex in pancreatic β cells"

Unit: G0 Cell Unit (Mitsuhiro Yanagida)

Title: Functional analysis of CCR4-NOT complex in pancreatic β cells

Abstract:

Regulation of mRNA decay in the cytoplasm is important for proper gene expression and its dysregulation causes various disorders. The carbon catabolite repression 4 (CCR4)–negative on TATA-less (NOT) complex (CCR4-NOT complex), a major deadenylase conserved in eukaryotes, catalyzes mRNA deadenylation which is the rate limiting step in mRNA decay pathway. By virtue of its deadenylation activity, it governs mRNA stability. Loss of subunits of the CCR4-NOT complex results in serious abnormalities in embryonic development and tissue function, suggesting that understanding the biological activities of this complex may pave the way for eventual development of therapeutics. Accordingly, this dissertation aimed at understanding the function of CCR4-NOT complex in pancreatic β cells by generating mice lacking the Cnot3 gene, which encodes an essential CCR4-NOT complex subunit, in β cells. Suppression of CNOT3 in β cells caused β cell dysfunction and diabetes. This was associated with the decreased expression of β cell-specific genes and increased expression of genes specifically repressed in β cells, called “β cell disallowed genes”. By combining whole transcriptome and mass spectrometry analyses and subsequent validations using quantitative real time PCR (qRT-PCR) and Western blotting, I found that mRNA and protein expression patterns were largely different from normal β cells upon CNOT3 suppression, which was clearly relevant to the observed phenotypes. I also found that some β-cell disallowed genes were stabilized in the absence of CNOT3, suggesting that their expression was maintained at low levels under the control of the CCR4-NOT complex. Together, this study uncovered mRNA deadenylation by CCR4-NOT complex as a novel molecular mechanism by which β cell identity and function are regulated. Next, in order to understand how the complex catalytic subunits contribute to the complex function, I used primary mouse embryonic fibroblasts (MEFs) prepared from mice lacking CCR4-NOT complex subunits. We found that maintenance of cell viability is one of the fundamental roles of the CCR4-NOT complex, which is mediated mainly by catalytic activity of the CNOT7/8 subunits. Their vital importance in regulating global mRNA expression is clearly reflected in the results of RNA-seq and bulk poly (A) tail assay. In contrast, CNOT6/6L subunits are dispensable for complex formation and MEFs viability.