Fig. 7: TUT1-mediated generation of the NCOR2-013 isoforms suppresses inflammatory response in T. marneffei-infected THP-1 macrophages.

a–d The human THP-1 macrophages were infected with T. marneffei conidia (MOI = 10) for 24 h. a Identification of potential RBPs that regulating AS events. rMATS was used to predict potential RBPs and then intersected with DEGs (T. marneffei-infected THP-1 macrophages vs. control cells). b The TUT1 motif map shows the regional binding of TUT1 in the flanking introns of skipped and included exon events with change in PSI > 20%, FDR < 0.05. The red, blue, and black lines represent the motif scores for the upregulated, downregulated, and control (non-regulated background) exons, respectively. c RT-qPCR and WB to detect the expression levels of TUT1 in T. marneffei-infected or uninfected THP-1 macrophages (n = 4 biological replicates, data are presented as mean values ± SD). d Visualization of origin genome profiles was performed with IGV. The black arrow represents the position of TUT1 motif, suggesting that it may bind between exon 14 and 15 of NCOR2. e RIP analysis was performed to verify the interaction of TUT1 and NCOR2 pre-mRNA. A Flag-tagged TUT1 was included as an indicator in the construction of TUT1 overexpressing macrophages. IP was performed with anti-Flag antibody, and RNA was subjected to agarose gel electrophoresis followed by RNA gel blot analysis (n = 4 biological replicates, data are presented as mean values ± SD). TUT1 overexpression (f) (n = 4 biological replicates, data are presented as mean values ± SD) and TUT1 knockdown (g) (n = 6 biological replicates, data are presented as mean values ± SD) in THP-1 macrophage were confirmed by RT-qPCR and WB. h–m THP-1 macrophages with TUT1 overexpression/knockdown and control cells were infected with T. marneffei (MOI = 10) for 24 h. The expression of NCOR2-013 was detected by RT-qPCR in T. marneffei-infected or uninfected TUT1 overexpressing THP-1 macrophages (h) and TUT1 knockdown THP-1 macrophages (i) (n = 3 biological replicates, data are presented as mean values ± SD). T. marneffei CFUs in TUT1 overexpressing THP-1 macrophages (j) (n = 12 biological replicates, data are presented as mean values ± SD) and TUT1 knockdown THP-1 macrophages (k) (n = 12 biological replicates, data are presented as mean values ± SD). The levels of TNF-α and IL-1β were detected by RT-qPCR in. T. marneffei-infected or uninfected TUT1 overexpressing THP-1 macrophages (l) and TUT1 knockdown THP-1 macrophages (m). GAPDH was used as the loading control (n = 3 biological replicates, data are presented as mean values ± SD). WB were shown were the representative blot. Two-tailed Student’s t test was used to determine significance, denoted by *(P < 0.05), **(P < 0.01), and ns (not significant). Supplementary material is available (Supplementary Fig. 6; Supplementary Data 1). MOI multiplicity of infection, NC negative control, TUT1 OE TUT1 overexpression, TUT1 RNAi TUT1 knockdown, Tm T. marneffei.