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

摘要

Background: Recent years have shown a marked increase in the use of next-generation sequencing technologies for quantification of gene expression (RNA sequencing, RNA-Seq). The expression level of a gene is a function of both its rate of transcription and RNA decay, and the influence of m RNA decay rates on gene expression in genome-wide studies of Gram-positive bacteria is under-investigated. Results: In this work, we employed RNA-Seq in a genome-wide determination of m RNA half-lives in the Gram- positive bacterium Bacillus cereus. By utilizing a newly developed normalization protocol, RNA-Seq was used successfully to determine global m RNA decay rates at the single nucleotide level. The analysis revealed positional degradation patterns, with m RNAs 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 m RNA decay rate correlates globally with m RNA expression level, GC content, and functional class of the ORF. Conclusions: To our knowledge, this study presents the first global analysis of m RNA decay in a bacterium at single nucleotide resolution. We provide a proof of principle for using RNA-Seq in bacterial m RNA decay analysis, revealing RNA processing patterns at the single nucleotide level.