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

Abstract

BackgroundRecent 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 mRNA decay rates on gene expression in genome-wide studies of Gram-positive bacteria is under-investigated.ResultsIn 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.ConclusionsTo 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.

Multiple Roles of RNase Y in Streptococcus pyogenes mRNA Processing and Degradation

“…The vast majority of mRNA half-lives increased in the ⌬rny mutant, though the stability profiles among the functional groups remained similar to what was observed in the WT. These observations support our hypothesis that mRNA stability is related to the function of the gene product and are in agreement with previous findings in other species (34,35).…”

“…These findings support the idea that RNA processing plays an important role in posttranscriptional regulation. The identification of processed RNAs on a genome-wide scale has been a challenge for traditional microarray analysis because RNA processing does not usually lead to an obvious change in mRNA abundance (35). However, since RNA processing changes the stability of mRNAs, those mRNAs that display segmental stability are very likely processed by some endoribonuclease(s).…”

Measurements of mRNA Degradation in Borrelia burgdorferi

“…mRNA lifetimes appear to be correlated with the function of the protein that they encode in E. coli (36), Lactococcus lactis (41), Prochlorococcus (39), and Bacillus cereus (42), although this correlation was not observed in B. subtilis (37). Generally, we observed rapid decay of transcripts encoding ribosomal proteins and RNA polymerase subunits in B. burgdorferi; however, rplQ and rpsO mRNA degradation was much slower than that observed for mRNAs encoding other ribosomal proteins ( Table 2).…”

Expression stability of 13 housekeeping genes during carbon starvation of Pseudomonas aeruginosa

“…However, quantification of prokaryotic mRNA is technically challenging, where the average half-life has been measured at 5.7 min for E. coli growing in M9 medium supplemented with glucose (Bernstein et al, 2002), and b 6 min in Gram-positive bacteria such as Bacillus cereus (Kristoffersen et al, 2012). Additionally, RNA extraction efficiency and differences in cell numbers present in the samples can introduce error in the method.…”