Fig. 2: RNase III-CLASH recovers RNA–RNA interactions in S. aureus JKD6009. | Nature Communications

Fig. 2: RNase III-CLASH recovers RNA–RNA interactions in S. aureus JKD6009.

From: RNase III-CLASH of multi-drug resistant Staphylococcus aureus reveals a regulatory mRNA 3′UTR required for intermediate vancomycin resistance

Fig. 2

a Histogram of the RNA classes recovered by RNase III-CLASH expressed as the number of hybrid reads (left) and number of unique RNA–RNA interactions (multiple hybrid reads can represent one RNA–RNA interaction) (right). b Distribution of the number of hybrid reads representing each RNA–RNA interaction recovered by RNase III-CLASH. Each RNA–RNA interaction type is indicated below and the number of independent experiments containing the interaction is indicated by the size of the data point. c Cumulative count of RNA–RNA interactions at start codons (indicated by the red dashed line). d Cumulative distribution function of RNA–RNA interaction strength (∆G, kcal/mol) for all RNA–RNA interactions recovered (left), or ncRNA–RNA interactions (right). Pairs of interacting RNAs were randomly shuffled and the distribution of interaction strength of randomly paired RNAs is shown in red. A two-sided Kolmogorov–Smirnov test was used to calculate p-values. e–h RNA–RNA interactions recovered by RNase III-CLASH are functional. Constitutively transcribed GFP translational fusions to SpoVG (e) and MgrA (f) were expressed in S. aureus RN4220 with or without transcription of cognate sRNAs (indicated below) and median fluorescence intensity (MFI) measured using flow cytometry. Histogram heights represent mean MFI and error bars represent the standard deviation (SD) from n = 3 biological replicates. Significance was calculated using a two-sided t-test. *p < 0.05. Predicted interactions between RNAs recovered in hybrid reads are indicated (right). Blue boxes indicate the ribosomal binding site and start codon for each mRNA. f Compensatory point mutations (M1) were introduced into mgrA and RsaA (indicated by arrows). MFI was measured for combinations of mgrA and RsaA M1 mutants (indicated below histogram). For interactions within the CDS, qRT-PCR was used to quantify target mRNA abundance (relative to gapA) in S. aureus RN4220 constitutively transcribing the sRNAs sRNA11 (g) or RNAIII (h) from the vector pICS3 (indicated below plot). The mRNAs agrA (g) and murQ (h) are expressed from the chromosomal loci. Histogram heights represent mean relative abundance and error bars indicate standard error from n = 3 biological replicates. p-values were calculated using a two-sided t-test. Predicted interactions between RNAs recovered in hybrid reads are indicated (right).

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