Necessity of HuR/ELAVL1 for the activation-induced cytidine deaminase-dependent decrease in topoisomerase 1 in...

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Activation-induced cytidine deaminase (AID) initiates antibody gene diversification via DNA cleavage.
• Efficient DNA cleavage requires an AID-dependent decrease in Topoisomerase 1 (Top1) levels, but the mechanism is unclear.
• HuR (ELAVL1) is an RNA-binding protein interacting with AU-rich elements.

Purpose of the Study:

• To elucidate the molecular mechanism by which HuR influences AID-dependent DNA cleavage.
• To investigate HuR's role in antibody gene diversification processes like class switch recombination (CSR) and somatic hypermutation (SHM).

Main Methods:

• Utilized HuR-knockout (KO) CH12 murine B lymphoma cells.
• Assessed CSR and SHM efficiencies, AID-dependent DNA cleavage levels, cell cycle, and Myc expression.
• Investigated the effect of reactive oxygen species (ROS) scavengers.
• Analyzed HuR binding to Top1 mRNA and Top1 protein synthesis.
• Performed Top1 knockdown rescue experiments in HuR-KO cells.

Main Results:

• HuR-KO CH12 cells exhibited reduced CSR and SHM efficiencies due to impaired AID-dependent DNA cleavage.
• HuR-KO cells showed normal cell cycles and Myc expression; ROS scavengers did not rescue CSR.
• HuR binds to Top1 mRNA, and its absence abolishes AID-dependent repression of Top1 protein synthesis.
• Top1 knockdown rescued CSR to IgG3 in HuR-KO cells, confirming Top1 accumulation as the cause of inefficiency.

Conclusions:

• HuR is critical for initiating antibody diversification and acquired immunity.
• HuR regulates AID-dependent DNA cleavage by repressing Top1 protein synthesis.
• The mechanism involves HuR's control over Top1 levels, impacting DNA cleavage efficiency in CSR and SHM.