Exploring the Functions of the Ccr4-NOT Complex and eIF4A2 in Cytosolic and ER Translation of Integrin Alpha 5, Integrin Beta 1, and Fibronectin 1

Abstract

The CCR4-NOT complex serves a critical function in mRNA deadenylation and subsequent degradation, significantly impacting post-transcriptional gene regulation. Its involvement in translational control, particularly in the context of specific mRNA targets, remains an area of active research. This study investigates the regulatory functions of the CCR4-NOT complex and its interactor eIF4A2 in cytosolic and endoplasmic reticulum (ER) translation processes, focusing on ITGA5, ITGB1, and FN1 mRNAs. Using qPCR, immunofluorescence, and ddPCR, we analyze mRNA distribution under various conditions, including eIF4A2 knockdown, rapamycin treatment, and CNOT1 knockdown. The experimental findings demonstrate that the CCR4-NOT complex exerts differential control over these transcripts, with its regulatory impact being highly context-dependent and varying substantially across diverse experimental parameters. Notably, Integrins alpha5 and beta1 mRNA levels showed significant changes upon CNOT1 knockdown, highlighting the complex's role in its regulation. The depletion of CNOT1, a key scaffolding component of the CCR4-NOT complex, disrupts the entire complex, affecting its overall function. This distinction is important when interpreting the results, as CNOT1 knockdown impacts the entire CCR4-NOT complex rather than just a single subunit. Additionally, our findings with RAB7 suggest increased degradation of FN1 mRNA, further emphasizing the complex's role in mRNA stability regulation. In conclusion, this study provides insights into the differential regulation of specific mRNAs by the CCR4- NOT complex, highlighting the importance of mRNA-specific features in determining susceptibility to CCR4-NOT-mediated regulation. These findings contribute to our understanding of post-transcriptional gene regulation mechanisms and their impact on protein expression.