Overlapping transcription and bacterial RNA removal

Abstract

The precise understanding of the biology of a living cell requires the identification and quantification of the molecular components necessary to sustain life. One such element is RNA. Two independent high-throughput strategies are available to identify the entire collection of RNA molecules produced by a cell population, which is currently known as the transcriptome. One technique relies on microarray technology (tiling arrays), whereas the second one relies on sequencing the RNA pool (RNA-seq) (1). Both techniques offer the advantage that the identification of the RNA content is not biased by protein-based genome annotation. The application of these methods to the transcriptome analysis in bacteria has uncovered the existence of a large amount of RNA molecules that overlap at least in some portion with protein-encoding RNA transcripts, generating perfect sense/antisense RNA duplexes (2⇓⇓–5). However, because transcriptome studies have been performed using microgram amounts of RNA purified from millions of bacterial cells instead of RNA purified from a single bacterium, the presence of overlapping sense/antisense RNAs from a genomic region does not necessarily mean that both sense and antisense transcripts are simultaneously present in the same bacteria. Hence, it might be possible that a subgroup in the bacterial population synthesized the sense transcript, another subgroup synthesized the antisense transcript, and consequently overlapping transcripts would never be together in the same cell. A report in PNAS by Lybecker et al. (6) provides clear evidences that both sense and antisense transcripts can be present simultaneously within the same bacterial cell. Using a monoclonal antibody that recognizes double-stranded RNA molecules (dsRNA) irrespectively of the nucleotide sequence, the authors perform immunoprecipitation assays to pull down dsRNA molecules (IP-dsRNA) from a total RNA sample extracted from Escherichia coli, and identified the purified dsRNA by RNA-seq.