Fig. 1: iRIL-seq charts global RNA-RNA interactome in hypervirulent K. pneumoniae. | Nature Communications

Fig. 1: iRIL-seq charts global RNA-RNA interactome in hypervirulent K. pneumoniae.

From: RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence

Fig. 1

a Schematic of in vivo RNA-RNA interactome profiling in HvKP. Interacting RNAs bound to Hfq are ligated by T4 RNA ligase 1, whose expression was induced from pBAD-t4rnl1 (pYC582) in live Klebsiella cells. Hfq-bound transcripts (singleton) and ligated fragments (chimeras) are enriched by Hfq-coIP and analyzed by deep sequencing. b Hfq represses hypermucoviscosity. Overnight cultures were centrifuged at 1000 g for 5 min. The OD600 of the supernatant was determined and normalized to the OD600 of starting culture. The acapsular i-wza mutant serves as a non-mucoid control. N = 3 biological replicates, bars indicate mean ± SD. **, p < 0.01; two-tailed Student’s t-test. c Western blot confirming Hfq pulldown. Strains carrying hfq−3xFLAG were grown to OD 3.0 and treated with 0.2% L-arabinose for 30 min. Lysates were subjected to coIP with anti-FLAG monoclonal antibody, or with mouse IgG as control. Lysates and IP samples are blotted using anti-FLAG, with GroEL as loading control. Image is representative of two experiments. Uncropped blots in Source Data. d Number of transcript types in significant chimeras (S-chimeras) for each combination of genomic elements. RNA1 and RNA2 constitute the 5’ portion and 3’ portion of a chimera, respectively. The numbers of interactions are indicated for each category in brackets. e Relative abundance of sRNAs detected in singleton and chimeric fragments. The percentage is obtained by dividing the number of fragments involving a given sRNA by the total fragments from sRNAs. The sRNA annotations are in Supplementary Data 2. f Circos plot showing the RNA-RNA interaction network in HvKP. Labeled sRNAs interact with >10 putative targets, and their interactions are shown in color. The number of interactions for each sRNA is indicated in brackets. A complete list of significant interactions is in Supplementary Data 4. Sequence motifs identified in RNA2 (g) and RNA1 (h, i) in S-chimeras. m, number of target sequences containing the motif; n, total number of target sequences. All motifs are in Supplementary Fig. 3.

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