Antisense RNAs (asRNAs) have been found in prokaryotes and eukaryotes, but whether they are biologically functional or merely byproducts of transcriptional read-through remains unknown. Now, a new methodology reported in the journal Proceedings of the National Academy of Sciences (1) could provide a way to answer this question. The high-throughput method for genome-wide identification of in vivo functional antisense RNAs in E. coli by deep sequencing was developed by Renée Schroeder's group at the University of Vienna.
As explained by Meghan Lybecker, lead author of the paper, the method is based on the assumption that functional asRNAs are present in cells as double-stranded RNAs (dsRNAs) duplexed with their target RNAs. The problem for Lybecker, though, was how to obtain these dsRNAs. At first, she struggled with digesting isolated RNA with single-strand-specific RNase to leave dsRNA.
“It’s very difficult to know when you’ve really gotten rid of all the single-stranded [RNA] and how much double-stranded [RNA] you have left. In all of those digests, you’re doing that at higher temperatures, so you could be really changing structures and losing and gaining double-stranded RNAs,” she explained.
Serendipitously, Lybecker encountered a paper presented at a journal club meeting describing a monoclonal antibody used to detect viral dsRNA in plants. “I thought, well, this is easier than what I’m doing now, and I’ll give it a shot since it hadn’t been done.” Using the J2 monoclonal antibody, which recognizes dsRNAs of at least 40 bp in length in a sequence-independent manner, Lybecker successfully immunoprecipitated and sequenced dsRNA from E. coli total RNA, performing cell lysis and RNA extraction at 4oC or on ice to prevent the artifactual formation of dsRNA.
dsRNA was immunoprecipitated from wild-type E. coli and a mutant strain lacking a functional enzyme in the dsRNA degradation pathway. cDNA libraries of Immunoprecipitated and total RNA from both the wild-type and mutant bacteria were deep-sequenced.
In the end, bioinformatic analyses revealed a total of 316 potential functional asRNAs; expression of 21 of these antisense/sense RNA pairs was verified by Northern blot analysis, strongly hinting that dsRNA plays a major role in E. coli gene regulation. While the precise function of asRNA remains an unsolved mystery for now, this new method opens the door for researchers to finally tackle the long standing question of asRNA function and reveal even more about the RNA world.
Lybecker, M., B. Zimmermann, I. Bilusic, N. Tukhtubaeva, and R. Schroeder. 2014. The double-stranded transcriptome of Escherichia coli. Proc Natl Acad Sci U S A. 2014. 111(8):3134-9. doi: 10.1073/pnas.1315974111