Fig. 3: KrrA directly binds to many RNA targets in vivo, including hitPRS.
From: An RNA-binding protein acts as a major post-transcriptional modulator in Bacillus anthracis
A To evaluate the RNA-binding ability of KrrA, a CLIP assay (UV crosslinkingg immunoprecipitation) was carried out in WT and ΔkrrA pOS1.PlgtkrrA-3xFLAG strains in the presence or absence of ‘205 treatment with or without UV crosslinkingg. The immunoprecipitated KrrA-3xFLAG protein was detected by Western blot using an anti-FLAG antibody (top panel). This serves as a control to evaluate the immunoprecipitation efficiency. The immunoprecipitated and radioactively labeled RNA-KrrA complexes were detected by phosphorimaging (bottom panel). The experiments were conducted at least three times and representative images are shown. Source data are provided as a Source Data file. B Overview of KrrA-RNA-binding profiles revealed by formaldehyde cross-linking RNA immunoprecipitation coupled with Illumina sequencing (fRIP-seq) under vehicle or ‘205-treated conditions. C Genes identified in the RIP-seq analysis with significantly increased enrichment grouped into predicted functional categories. D Volcano plot showing KrrA-binding RNA transcripts enriched following ‘205 treatment compared to vehicle. Transcripts that were most significantly enriched upon ‘205 treatment are highlighted in blue. The log2(fold change) cutoff is ≥2 and the P value cutoff is <0.05, both of which are indicated by gray lines. E A zoom-in example of KrrA binding to the hitPRS transcripts identified by fRIP-seq. Two biological replicates were included for each condition. S/N denotes the signal-to-noise ratio for peak calling. F KrrA specifically binds to hitR transcript under vehicle conditions and transcripts of the entire hitPRS operon upon ‘205 treatment. ND not detectable. G KrrA binding to the transcripts of hitPRS was confirmed by fRIP-qPCR under vehicle or ‘205-treated conditions. The gene gyrA was used as a control. The data are expressed as the mean ± SD (n = 3). Significant differences are determined by two-tailed t tests. Source data are provided as a Source Data file.
