Fig. 1: RBPome capture in S. aureus. | Nature Communications

Fig. 1: RBPome capture in S. aureus.

From: The RNA-bound proteome of MRSA reveals post-transcriptional roles for helix-turn-helix DNA-binding and Rossmann-fold proteins

Fig. 1

a S. aureus 2C and PTex RBPome capture workflows. See ‘Methods’ section for a detailed description of the approaches. CL cross-linked, 2C complex capture, PTex phenol–toluol extraction, aq aqueous, inter interphase, org organic. b Western blot and silver staining analysis of the USA300 RNase III-HTF 2C experiments. The UV irradiation doses used (J/cm2) are indicated. RNase III-HTF (black triangle) was detected using anti-FLAG antibodies. ‘Input’ indicates µg of total lysate loaded on the gel. The black asterisk indicates the Benzonase used to degrade the RNA. Non-cross-linked cells were used as negative controls. TSB tryptic soy broth, LPM low phosphate medium pH 7.5. c Western blot results and Coomassie brilliant blue staining of the USA300 RNase III-HTF PTex experiments, performed with the indicated UV intensities (J/cm2) in LPM medium. One percent of the total lysate was loaded for comparison. Original images for the experiments shown in b, c are provided in the Source data. d Volcano plots of proteins enriched in TSB (left) and LPM medium 2C data (right) (n = 6; two independent biological replicates, three technical replicates). P values were generated by empirical Bayes moderated t test in limma and adjusted by Benjamini–Hochberg method. Proteins with a log-fold change >2 and a −log10 adjusted P value of ≥1.3 (indicated with dashed lines) were considered significantly enriched (red dots). The total number of proteins significantly enriched in the cross-linked samples is indicated. e As in d but now for the PTex results (3 biological replicates) from cells grown in LPM medium. An adjusted P value threshold of 0.05 was used to select proteins enriched in the cross-linked samples. f Overlap of significantly enriched RBPs identified in TSB and LPM 2C data. g Overlap of JDK6009 and USA300 RBPs identified by 2C and PTex in cells grown in LPM medium. h Overlap of USA300 RBPs identified by 2C with published E. coli OOPS and TRAPP RBPome data sets. i Overlap of USA300 RBPs identified by 2C with RBPs predicted by TriPepSVM and RBPPred. TRAPP total RNA-associated protein purification, OOPS orthogonal organic phase separation.

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