The double-stranded transcriptome of Escherichia coli

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• High-throughput transcriptome analyses have identified numerous bacterial antisense RNAs (asRNAs).
• The functional significance and quantity of these asRNAs remain largely uncharacterized.
• Antisense RNA mechanisms often involve RNA-RNA interactions, suggesting duplex formation.

Purpose of the Study:

• To develop and implement a genome-wide, high-throughput method for identifying functional asRNAs in vivo.
• To investigate the role of double-stranded RNA (dsRNA) in bacterial gene regulation.
• To characterize the locations and prevalence of functional asRNAs in Escherichia coli.

Main Methods:

• Development of a method to isolate dsRNAs using dsRNA-specific antibody immunoprecipitation.
• Deep sequencing of total RNA and immunoprecipitated dsRNA from wild-type and RNase III mutant E. coli strains.
• Application of a statistical model to identify biologically relevant dsRNA regions and their genomic locations.

Main Results:

• Identification of 316 potentially functional asRNAs in the RNase III mutant strain.
• Functional asRNAs were predominantly located opposite the 5' ends of transcripts, but also near ncRNAs, gene junctions, and 3' ends.
• Twenty-one sense/antisense RNA pairs were confirmed by Northern blot, with altered RNA levels in the RNase III mutant.

Conclusions:

• A significant amount of dsRNA is formed within E. coli cells.
• RNase III plays a crucial role in degrading or processing these dsRNAs.
• dsRNA formation and processing are integral to bacterial gene regulation.