SLAM-seq analysis of WT and TTP-KO basophils treated with antigen/IgE stimulation
Basophils play crucial roles in type 2 immune responses, such as chronic allergic inflammation and protective immunity against parasites. However, the molecular mechanisms underlying the regulation of proinflammatory molecule production by basophils remain unclear. To investigate the role of RNA-binding proteins (RBPs), we first analyzed the gene expression of CCCH zinc finger proteins in antigen/IgE-stimulated basophils and identified that Zfp36, encoding tristetraprolin (TTP), was the most significantly upregulated gene encoding a CCCH zinc finger protein. To explore the functions of TTP in basophils, we generated TTP-knockout (TTP-KO) mice using the CRISPR-Cas9 method and conducted bulk RNA-seq analysis of antigen/IgE-stimulated basophils from wild-type (WT) and TTP-KO mice. TTP-KO basophils exhibited elevated mRNA expression of pro-inflammatory mediators, such as Il4, Il13, Areg, Ccl3, and Cxcl2, compared to WT basophils, suggesting the key role of TTP in the expression of mRNAs encoding proinflammatory mediators. Since TTP is known to regulate mRNA stability in other immune cell types, we evaluated the mRNA stability of WT and TTP-KO basophils under antigen/IgE stimulation using SLAM-seq [thiol (SH)-Linked Alkylation for the Metabolic sequencing of RNA] analysis. SLAM-seq analysis revealed that the mRNA half-lives of proinflammatory molecules, such as Il4, Il13, Areg, Ccl3, and Cxcl2, were prolonged by TTP deficiency, suggesting the importance of TTP in regulating the stability of mRNAs encoding inflammatory molecules in basophils. Overall design: To examine the effect of TTP deficiency on mRNA stability in antigen/IgE-stimulated basophils, WT and TTP-KO basophils sensitized with hapten TNP-specific IgE were first treated with TNP-conjugated OVA for 1.5 hours in the presence of 4-thiouridine (s4U), then incubated for 0, 1, 2, and 4 hours in the absence of TNP-OVA and s4U, and subjected to alkylation reaction and RNA-seq analysis. The half-life of each mRNA in WT and TTP-KO basophils was calculated using the GRAND-SLAM and grandR packages.