Fig. 6: Analysis of the CsrA foci associated RNAs.

A The RNA content of the CsrA foci. EPEC expressing CsrA-FLAGx3-GFP or wild-type EPEC expressing untagged CsrA (negative control) were grown to OD 0.6 and subjected to the foci-enrichment protocol (Fig. S9), followed by RNA extraction and library preparation. RNA-seq was performed and the sequenced RNA was mapped to the EPEC genome. The experiment was performed in three biological repeats and gene-assigned read counts from the six sequenced libraries were normalized and compared by DESeq2⁵⁴. DESeq2 reports for each of the compared genes the Log₂ fold change (Log₂FC) of the normalized read counts between the CsrA foci and the wt control along with the p-value corrected for multiple hypothesis testing (padj) (Supplementary Data 3). For a gene to be considered statistically significantly enriched in the CsrA foci we applied stringent filtering requiring Log₂FC > 2 and padj<10⁻⁴. The genes that passed this stringent filtering are shown here with their respective Log₂FC (x-axis) and –Log₁₀ (padj) (y-axis) values. The identity of some of the genes and their biological function (manually curated from the literature) is indicated by color coding. RNAs belonging to the LEE1 (Ler, EscE) and LEE7 (GrlA) mRNAs are highlighted in bold fonts. B Representation of the different categories within the foci. A pie chart displays the number of genes detected in the CsrA foci assigned to the different categories. Genes were categorized as in (A). C mRNAs associated with the CsrA foci of EPEC compared to CsrA-associated RNAs in E. coli K12. A Venn diagram showing the genes obtained from the EPEC foci transcriptome (green circle) and E. coli K12 CsrA CLIP-seq analysis³³ (red circle). Only genes common to the two strains were included in this comparison. The mRNAs were defined by their CDS boundaries. D sRNAs associated with the CsrA foci of EPEC compared to CsrA-associated sRNAs in E. coli K12. Like in (C), only sRNAs common to EPEC and E. coli K12 are compared. All of the sRNAs detected in the CsrA focus, but AspX, are known to contain two or more verified or putative CsrA binding sites (i.e., GGA triplet sequence). All of the sRNAs detected only in the CLIP-seq, but GcvB, contain one or no CsrA binding site³³.