Global mRNA decay analysis at single nucleotide resolution reveals segmental and positional degradation patterns in a Gram-positive bacterium

In this work, we employed RNA-Seq in a genome-wide determination of mRNA half-lives in the Gram-positive bacterium Bacillus cereus. By utilizing a newly developed normalization protocol, RNA-Seq was used successfully to determine global mRNA decay rates at the single nucleotide level. The analysis revealed positional degradation patterns, with mRNAs being degraded from both ends of the molecule, indicating that both 5' to 3' and 3' to 5' directions of RNA decay are present in B. cereus. Other operons showed segmental degradation patterns where specific ORFs within polycistrons were degraded at variable rates, underlining the importance of RNA processing in gene regulation. We determined the half-lives for more than 2,700 ORFs in B. cereus ATCC 10987, ranging from less than one minute to more than fifteen minutes, and showed that mRNA decay rate correlates globally with mRNA expression level, GC content, and functional class of the ORF.To our knowledge, this study presents the first global analysis of mRNA decay in a bacterium at single nucleotide resolution. We provide a proof of principle for using RNA-Seq in bacterial mRNA decay analysis, revealing RNA processing patterns at the single nucleotide level.The expression of a gene is subject to numerous levels of regulation; from the initiation of transcription, via RNA processing, translation and degradation of the message, and finally through processing and degradation of the protein product itself. During the past years, it has become apparent that, in addition to transcription initiation, alteration in the rate of messenger decay constitutes an important factor in the regulation of a gene's steady state mRNA expression level [1-4]. In bacteria, several examples are known of altered decay rates for specific mRNAs following changes in cell growth conditions, often mediated by small non-coding RNAs (for examples, see [3-7]). Although the vast majority of Escherichia coli mRNAs were reported not to exhibit a highly va