Extended Data Fig. 6: Examples of identified nested antisense RNAs.

B. subtilis shows a number (n = 35, see Methods for selection criteria) of mRNAs with long untranslated regions fully encompassing genes in the antisense directions, which we call nested antisense RNAs (also termed non-contiguous operons⁵³ or excludons⁵⁴). The majority (n = 29/35) of these have a fold-change in mRNA level less than twofold upon rho deletion (Fig. 3b). a, Schematic of a nested antisense RNA with corresponding Rend-seq signal, with orange peaks and blue peaks marking 5′ and 3′ boundaries of the transcript. b, Representative examples of nested antisense RNAs with mRNA level fold change upon rho deletion less than 2. Rend-seq data (peak shadows removed, see³⁵ for details on data processing) is shown. Orange and blue signal correspond to summed 5′-mapped reads and 3′-mapped reads, respectively (rpm: reads per million). Top trace corresponds to wild type, and bottom trace to ∆rho. Horizontal size marker provides positional scale (200 bp) on each subpanel. Sense and antisense genes are shown in dark and light grey, respectively. Double line breaks (//) indicate truncated Rend-seq signal at peaks. Dashed lines mark regions for which fold-change in read density for ∆rho/WT was estimated. The fold-change for each instance is indicated on the graph. c, Same as b, with representative examples of nested antisense RNAs with increased expression upon rho deletion (Fig. 3b). Three nested antisense RNAs were found in E. coli with identical criteria. See Supplementary Data 3 for a list of nested antisense RNAs identified.

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