Fig. 5: ILF3 enhanced the production of two vsiRNAs. | Nature Communications

Fig. 5: ILF3 enhanced the production of two vsiRNAs.

From: Identification of antiviral RNAi regulators, ILF3/DHX9, recruit at ZIKV stem loop B to protect against ZIKV induced microcephaly

Fig. 5

Distribution of vsRNA reads (20–23 nt in size) in the positive- and negative-stranded ZIKV 5′UTR genome from total small RNA seq (A) or AGO-IP small RNA seq (B), respectively. The relative abundances of positive- and negative-stranded vsRNAs are indicated. C Northern Blot validated the expression of vsiR-1 and vsiR-2. n = 2 biological replicates/group. D RT-qPCR analysis of ZIKV vsiR-1 and vsiR-2 expression in ZIKV-infected control or ILF3-knockdown SK-N-BE(2) cells (MOI = 1) at 48 hpi. Bars, mean ± SD, n = 3 biological replicates/group, two-tailed Student’s t-test. ***p ≤ 0.001. RT-qPCR analysis of ZIKV vsiR-1 and vsiR-2 expression in ZIKV-infected control or ILF3-knockdown brain organoid (E) or Nestin-Cre ILF3-cKO mice (F). Bars, mean ± SD, n = 3 biological replicates/group, two-tailed Student’s t-test. *p < 0.05, **p < 0.01. G Western blot showing the interactions with ILF3, DICER, and AGO proteins in ILF3 endogenous IP from mock, ZIKV MR766, or PRVABC.59-infected cells (MOI = 1) at 48 hpi. n = 3 biological replicates/group. H PrismNET illustrates the enrichment of ILF3 at 5′UTR of ZIKV vRNA. Upper panel, two heatmap tracks display sequence response and structure response respectively; red color showing highly attention region. Bottom panel red color illustrates the enrichment position of ILF3 at ZIKV 5′UTR vRNA. I RIP-qPCR shows the enrichment of ILF3, DICER, and AGO2 at SLB and cHP region of ZIKV vRNA in ZIKV MR766-infected SK-N-BE(2) cells. Five primer pairs were used to assess the enrichment across ZIKV vRNA. Bars, mean ± SD, n = 3 biological replicates/group, two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p ≤ 0.001. J RNA EMSA was performed using purified DICER and ILF3 protein as indicated and probed with biotin-labeled ZIKV 5′UTR RNA or ZIKV 5′UTR mutant RNA (M1B). n = 2 biological replicates/group. K DICER processing assay shows vsiRNA cleavage ability with DICER, ILF3 along or DICER and ILF3 together to biotin-labeled ZIKV 5′UTR RNA or ZIKV 5′UTR mutant RNA (M1B) respectively. n = 2 biological replicates/group. Source data are provided as a Source Data file.

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