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In our recent article, “Widespread Antisense Transcription in Escherichia coli” (1), we describe the identification of ~1,000 antisense RNAs (aRNAs) in the model bacterium Escherichia coli. This finding is significant because it suggests that aRNAs represent a major class of regulators in bacteria. Slonczewski expressed concerns regarding (i) the documentation of methods used, (ii) possible contamination of the cDNA library, (iii) insufficient controls, and (iv) failure to consider alternative interpretations of the data (4).
Due to strict space limitations for Observations, we provided only the most relevant aspects of the methods used. A detailed description of the library construction is included at the end of this letter.
Library construction involved ligation of an RNA linker, reverse transcription, and two rounds of nested PCR. This method effectively eliminates the possibility of contamination by exogenous DNA or RNA. We attempted to construct a control cDNA library from a mock sample lacking RNA. We cloned and sequenced several individual fragments from this library, all of which were products formed by ligation of multiple mutant RNA linker oligonucleotides. Hence, we did not subject this library to high-throughput sequencing. This observation supports the idea that contamination by exogenous DNA or RNA was not a concern.
As described in the article, we validated the aRNAs using bioinformatic and experimental approaches. Crucially, we demonstrated that (i) putative aRNAs are significantly enriched for “A” at the initiating nucleotide (P < 1e−50) and are indistinguishable in this respect from published RNA 5′ ends (P = 0.40); (ii) −10 hexamers for putative aRNAs have significantly better matches to the consensus sequence than random sequences do (P = 8.8E−102) and are indistinguishable from those of published RNAs (P = 0.49); (iii) 9 of 10 putative aRNA promoters tested resulted in significant expression of a lacZ reporter gene; in all nine cases, expression was dependent upon an intact −10 hexamer, indicative of a functional promoter; and (iv) one of two putative aRNAs tested repressed expression of the overlapping protein-coding gene.
Our data are most easily explained by the presence of a large number of aRNAs and are not consistent with any other reasonable interpretation. We note that no other possible interpretations and no other controls were suggested by Slonczewski.
Lastly, our conclusions are consistent with those of many other studies. In particular, a study in which the authors identified a similar number of aRNAs in Helicobacter pylori (2) was published while our work was in press.
For cDNA library construction, MG1655 E. coli cells were grown in LB to an optical density at 600 nm (OD600) of 0.7. RNA was purified using the hot-phenol method (3) and treated with DNase I. rRNA was removed by two rounds of treatment with the MICROBExpress kit (Ambion). RNA was treated with tobacco acid pyrophosphatase (TAP). TAP-treated RNA was purified by phenol extraction and ethanol precipitation. An RNA oligonucleotide (5′-ACACUCUUUCCCUACACGACGCUCUUCCGAUCU-3′) was ligated to the RNA 5′ ends using T4 RNA ligase 1 (NEB) for 1 h at 37°C. RNA was gel purified to remove unligated 5′ linker oligonucleotide. RNA was reverse transcribed with 5′-GTTTCCCAGTCACGATCNNNNNNNNN-3′ and SuperScript III reverse transcriptase (Invitrogen). cDNA was amplified by PCR (25 cycles) using primers 5′-GTTTCCCAGTCACGATC-3′ and 5′-ACACTCTTTCCCTACACG-3′. DNAs of lengths between 80 and 200 bp were gel purified and reamplified by PCR using primers 5′-GTTTCCCAGTCACGATC-3′ and 5′-ACGCTCTTCCGATCT-3′. One-hundredth of the DNA sample was amplified by PCR using the primers 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′ and 5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGTTTCCCAGTCACGATC-3′.
Citation Wade, J. T., J. E. Dornenburg, A. M. DeVita, and M. J. Palumbo. 2010. Reply to “Concerns about recently identified widespread antisense transcription in Escherichia coli.” mBio 1(2):e00119-10. doi:10.1128/mBio.00119-10.