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Functional characterization of the disease-associated CCL2 rs1024611G-rs13900T haplotype: The role of the RNA-binding

Feroz Akhtar1, Joselin Hernandez Ruiz2, Ya-Guang Liu3

  • 1Department of Health and Behavioral Sciences, Texas A&M University- San Antonio, Texas, USA.

Biorxiv : the Preprint Server for Biology
|November 14, 2023
PubMed

Insights

Genetic variations in the CCL2 gene, specifically rs13900, influence its expression by affecting mRNA stability and translation. This impacts monocyte recruitment and disease susceptibility, with the T allele linked to increased CCL2 expression via HuR binding.

Area of Science:

  • Genetics
  • Molecular Biology
  • Immunology

Background:

  • CC-chemokine ligand 2 (CCL2) drives monocyte/macrophage recruitment in diseases like atherosclerosis and HIV-associated neurocognitive disorder (HAND).
  • The CCL2 rs1024611 polymorphism is linked to increased CCL2 expression and adverse disease outcomes, but its molecular mechanism is unclear.
  • The rs13900 polymorphism in the CCL2 3' UTR is in perfect linkage disequilibrium with rs1024611 and has been shown to cause allelic expression imbalance.

Purpose of the Study:

  • To investigate whether the rs13900 polymorphism modulates CCL2 expression by altering mRNA turnover or translatability.
  • To elucidate the molecular mechanisms underlying the association between rs13900 and differential CCL2 expression.
  • To determine the role of the RNA-binding protein Human Antigen R (HuR) in mediating allele-specific CCL2 expression.

Main Methods:

  • Reporter vector assays to assess allele-specific activity and translatability.
  • In vitro and ex vivo binding assays to evaluate the interaction of rs13900 alleles with HuR.
  • mRNA stability assays to compare the stability of CCL2 transcripts carrying different rs13900 alleles.
  • Overexpression and silencing studies to investigate HuR's role in allele-specific CCL2 expression.
  • Transcriptomic analysis to identify gene expression signatures associated with specific CCL2 genotypes.

Main Results:

  • The rs13900 T allele conferred greater stability to the CCL2 transcript compared to the C allele.
  • The rs13900 T allele exhibited increased binding to HuR in vitro and ex vivo.
  • Alleles of rs13900 demonstrated differential activity in reporter vectors, influencing reporter transcript translatability.
  • HuR was confirmed to play a role in mediating allele-specific effects on CCL2 expression.
  • The rs1024611G-rs13900T genotype was associated with a distinct transcriptomic signature related to inflammation and immunity.

Conclusions:

  • The rs13900 polymorphism influences CCL2 expression through allele-specific modulation of mRNA stability and translatability.
  • Differential binding of HuR to the rs13900 polymorphism is a key mechanism underlying allele-specific CCL2 expression.
  • These findings provide a molecular basis for interindividual differences in CCL2-mediated disease susceptibility.

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