Overlapping transcription and bacterial RNA removal.

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. Previous studies have identified examples of at least four different mechanisms to generate dsRNA duplexes in bacteria (2, 7): (i) the presence of bona fide noncoding antisense RNAs (asRNA); (ii) overlapping in the 5′ region of mRNAs from contiguous genes that are transcribed in divergent directions; (iii) overlapping in the 3′ regions of mRNAs from contiguous genes transcribed in convergent directions; and finally, (iv) genes that, being located in the middle of an operon, are transcribed in the opposite direction to the rest of the operon. According to these mechanisms, the entire mRNA molecule seems to be susceptible to be targeted by an overlapping transcript. However, with only a handful of transcriptomes available so far, it is too early to establish whether overlapping transcription preferentially locates in a specific region of the mRNA relative to the ORF. In this respect, Lybecker et al. find that the majority of IP-dsRNAs correspond to the 5′ region of genes (50%), whereas only 0.5% of the IP-dsRNAs correspond to overlapping transcripts that affect the 3′ region (6). The most common scenario is overlapping between long 5′UTRs of divergently transcribed genes, followed by overlapping caused by asRNAs transcribed opposite to the 5′/intergenic ends. This description of such a strong bias of dsRNAs toward the 5′ region is unique. Whether differences in the size of overlapping regions might determine a more efficient 5 ́ 3 ́ 5 ́ 3 ́