Antisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. The ectopic expression of plant Tombusvirus p19 in Escherichia coli stabilizes ∼21-nucleotide (nt) dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA. RNase III-produced small dsRNA were formed at most bacterial genes in the bacterial genome and in a plasmid. We classified the types of asRNA in genomic clusters producing the most abundant p19-captured dsRNA and confirmed RNase III regulation of asRNA and sense RNA decay at three type I toxin-antitoxin loci and at a coding gene, rsd. Furthermore, we provide potential evidence for the RNase III-dependent regulation of CspD protein by asRNA. The analysis of p19-captured dsRNA revealed an RNase III sequence preference for AU-rich sequences 3 nucleotides on either side of the cleavage sites and for GC-rich sequences in the 2-nt overhangs. Unexpectedly, GC-rich sequences were enriched in the middle section of p19-captured dsRNA, suggesting some unexpected sequence bias in p19 protein binding. Nonetheless, the ectopic expression of p19 is a sensitive method for identifying antisense transcripts and RNase III cleavage sites in dsRNA formed by overlapping sense and antisense transcripts in bacteria.

中文翻译：

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Tombusvirus p19捕获大肠埃希氏菌中有义和反义转录物重叠形成的RNase III切割的双链RNA。

反义转录在细菌中很普遍。通过与重叠的有义RNA进行碱基配对，反义RNA（asRNA）可以形成双链RNA（dsRNA），并被dsRNA内切核糖核酸酶RNase III切割。植物Tombusvirus p19在大肠杆菌中的异位表达稳定了约21个核苷酸（nt）dsRNA RNase III降解中间体，这使我们能够通过对p19捕获的dsRNA进行深度测序来表征原本高度不稳定的asRNA。RNase III产生的小dsRNA在细菌基因组和质粒中的大多数细菌基因上形成。我们在产生最丰富的p19捕获的dsRNA的基因组簇中对asRNA的类型进行了分类，并确认了RNase III对asRNA的调节，并在三个I型毒素-抗毒素基因座和一个编码基因处检测了RNA的衰减，rsd。此外，我们为asRNA提供了对CspD蛋白的RNase III依赖性调控的潜在证据。对p19捕获的dsRNA的分析表明，RNase III序列优先于切割位点两侧的3个核苷酸的AU富集序列和2 nt突出端的GC富集序列。出乎意料的是，富含GC的序列在p19捕获的dsRNA的中间部分富集，表明p19蛋白结合中存在一些意想不到的序列偏向。尽管如此，p19的异位表达是鉴定细菌中有义和反义转录物重叠形成的dsRNA中反义转录物和RNase III切割位点的灵敏方法。