RNase III-Binding-mRNAs Revealed Novel Complementary Transcripts in Streptomyces

Abstract

cis-Antisense RNAs (asRNAs) provide very simple and effective gene expression control due to the perfect complementarity between regulated and regulatory transcripts. In Streptomyces, the antibiotic-producing clade, the antisense control system is not yet understood, although it might direct the organism's complex development. Initial studies in Streptomyces have found a number of asRNAs. Apart from this, hundreds of mRNAs have been shown to bind RNase III, the double strand-specific endoribonuclease. In this study, we tested 17 mRNAs that have been previously co-precipitated with RNase III for antisense expression. Our RACE mapping showed that all of these mRNAs possess cognate asRNA. Additional tests for antisense expression uncovered as-adpA, as-rnc, as3983, as-sigB, as-sigH, and as-sigR RNAs. Northern blots detected the expression profiles of 18 novel transcripts. Noteworthy, we also found that only a minority of asRNAs respond to the absence of RNase III enzyme by increasing their cellular levels. Our findings suggest that antisense expression is widespread in Streptomyces, including genes of such important developmental regulators, as AdpA, RNase III, and sigma factors.

Keywords: RNase III; Streptomyces; antibiotics; cis-antisense RNA; gene expression control.

Figure 1

Novel asRNAs revealed by 5′and 3′RACE method, and their genome locations (sequence of asRNA in red, sequence of mRNA in blue). Transcriptional start sites are indicated by arrows. Full green line represents 5′RACE inner primers, dotted green line represents 5′RACE outer primers, full orange line represents 3′RACE inner primers, dotted orange line represents 3′RACE outer primers. (A) Experimental set; (B) Control set; (see text for details).

Figure 2

Differential expression analyses of novel asRNAs and their target mRNAs in WT and rnc strains. Three black lines (from long to short) represent RNA samples from vegetative mycelium (24 h), aerial mycelium (48 h), and spores (72 h), respectively. To enable comparison of the expression profiles, all asRNA-mRNA pairs from both WT and rnc strains were analyzed on the same blot. Sizes of the products well corresponded to those obtained by RACE. Primers used are shown on the right. The 5S loading control is included below. The signal quantification is presented in Table S2. (A) Experimental set; (B) Control set; (see text for details).