RsaE processing, stability and interaction with <i>sucC</i> mRNA.

(A) Quantification of full-length and processed RsaE (RsaEp) transcripts in PS2 and PS10 by dRNA-seq analysis, with (+) and without (-) TEX treatment. CPM: counts per million reads, calculated as NOAR (number of aligned reads)*106/TNOAR (total number of aligned reads). Black columns indicate the number of transcription starts of full-length RsaE (100 nt); the number of processed RsaE species (RsaEp, 76–78 nt) are shown as white, light grey and dark grey columns, respectively. (B) Stability determination of full-length (RsaE) and processed (RsaEp) RsaE species. S. epidermidis PS2 was grown in TSB to early-exponential growth stage and RNA was isolated before (0) and at the time points indicated after transcription blocking by rifampicin. RNA samples were subjected to Northern blot analyses using radioactively labeled ssDNA-oligonucleotide probes targeting either RsaE or 5S rRNA as loading control. (C) Proposed secondary structure of the processed RsaE species (RsaEp, 78 nt) according to mFold-4.7-based prediction [65]. Grey boxes highlight positions of the C-rich motifs within the processed RsaE molecule. Nucleotides complementary to the antisense-RsaE RNA probe used in (D) are underlined. Arrows indicate alternative RsaE processing sites identified by dRNA-seq. (D) EMSAs using increasing amounts of sucC target RNA and radioactively labeled (*) full-length (RsaE, left panel) or processed RsaE (RsaEp, right panel) as binding partners. (E) sucC/RsaEp EMSA upon competition with an antisense-RsaE RNA oligonucleotide or unspecific tRNAs during complex formation. 200 nM of sucC target RNA was mixed with increasing amounts of antisense-RsaE RNA oligonucleotide (lanes 3–6) or a 500-fold excess of yeast tRNAs (lane 7) prior to addition of 200 nM radioactively (*) labeled RsaEp to the samples. Filled triangles in (D) and (E) indicate labeled unbound RsaE and open triangles mark RsaE/target complexes.