Genomic Analysis of the Role of RNase R in the Turnover of Pseudomonas putida mRNAs

Abstract

RNase R is a 3-5 highly processive exoribonuclease that can digest RNAs with extensive secondary structure. We analyzed the global effect of eliminating RNase R on the Pseudomonas putida transcriptome and the expression of the rnr gene under diverse conditions. The absence of RNase R led to increased levels of many mRNAs, indicating that it plays an important role in mRNA turnover.Attaining a proper balance between mRNA synthesis and degradation is essential for determining levels of gene expression. The degradation of mRNAs in eubacteria involves the actions of different RNases and usually starts with endonucleolytic cleavage of the RNA molecule, followed by exoribonucleolytic digestion of the resulting fragments to yield 5Ј mononucleotides (21). RNA decay has been studied mainly in Escherichia coli, which has at least five endonucleases and eight exoribonucleases (21). Among the 3Ј-5Ј exoribonucleases, RNase II and polynucleotide phosphorylase (PNPase) are responsible for most mRNA degradation (24). Some of these nucleases form part of the degradosome, a complex devoted to RNA turnover. In E. coli, PNPase forms part of the degradosome while RNase II does not (reference 37 and references therein). Both nucleases are significantly inhibited by RNA secondary structures; the action of PNPase is probably helped by RhlB RNA helicase, which is also part of the degradosome. RNase R is a 3Ј-5Ј exoribonuclease that is very processive and can efficiently digest RNAs having extensive secondary structures, such as rRNA, RNAs containing repetitive extragenic palindromic (REP) sequences, or the tmRNA (transfer-messenger RNA) required for trans-translation (7,8,33). RNase R is also involved in the processing of 16S and 5S rRNA (30). The gene encoding RNase R has been named vacB or rnr. In E. coli, rnr gene mutant strains are viable under standard laboratory conditions, although a double mutant lacking both RNase R and PNPase is not viable (8,9). This suggests that PNPase compensates for the absence of RNase R in the rnr mutant strains. The levels of RNase R increase in response to stress conditions, such as entry into stationary phase, cold shock, or growth under carbon, nitrogen, or phosphorus limitation (5, 6), suggesting that it performs an important role in vivo. Homologues of the E. coli rnr gene are found in most bacteria (22,41), which supports the notion that RNase R plays an important role in the metabolism of at least some kinds of RNAs. In Shigella flexneri (38) and Aeromonas hydrophyla (15), inactivation of rnr reduces pathogenicity.Although many of the conclusions about RNA decay obtained for E. coli are valid for other bacterial species, variations may exist related to the different lifestyles of the microorganisms. For example, RNase R forms part of the degradosome in Pseudomonas syringae, but not in E. coli (29). RNase R may become particularly important at low temperatures, since inactivation of the rnr gene inhibits growth of P. syringae (30) and Pseudomonas putida (32) at 4°C, but not at 22°C. At low temper...

Loss of RNase R Induces Competence Development in Legionella pneumophila

“…The Tn903dIIlacZ insertion has been mapped at nucleotide (nt) 513 of the RNase R coding sequence and does not produce a translational fusion with lacZ. Since RNase R has been reported for several species to be required for growth at low temperatures (5,16,17,26), we analyzed the growth of an L. pneumophila rnr strain under axenic conditions on solid rich medium at optimal and suboptimal growth temperatures. The RNase R mutant did not show significant growth defects at optimal (37°C) or elevated (42°C) growth temperatures (Fig.…”

“…Instead, we found that the comEA mRNA has a longer half-life in the rnr mutant, indicating that it is a substrate of RNase R. Indeed, the sole inactivation of the exoribonuclease activity of RNase R at 30°C is sufficient to induce competence development. A recent genome-wide analysis of mRNA decay in an rnr mutant of Pseudomonas putida has revealed the important role of RNase R in mRNA turnover (17). Although RNase R is active on mRNAs containing REP sequences, the mRNAs affected by the lack of RNase R were not enriched in REPcontaining sequences.…”

“…Consistent with the notion of the involvement of RNase in the degradation of RNA being stabilized by low temperature, RNase R has been found as part of the degradosome in the cold-adapted strain Pseudomonas syringae Lz4W (25). RNase R is also essential for growth at low temperatures for Pseudomonas syringae, Pseudomonas putida, and Aeromonas hydrophila (16,17,26). In the reduced-genome intracellular pathogen Mycoplasma genitalium, RNase R appears to be the only exoribonuclease (22) and is also thought to be essential, since the gene could not be interrupted (20).…”

Transcriptome Dynamics of Pseudomonas putida KT2440 under Water Stress

“…To partially validate the trends of the microarray results, quantitative reverse transcription-PCR (qRT-PCR) was performed on isolated RNA (obtained from a batch of [P]PSMs separate from the ones used for the microarray) for selected genes (algT, alg8, mucA, fliE, and flgM) with 3 replicates for each condition (4 h, 72 h, and control). The primer sets (13,22) are listed in Table S3 in the supplemental material. cDNA synthesis and amplification were performed using the Qscript 1-Step Sybr green qRT-PCR kit (Quanta Biosciences, Maryland) in a Chromo4 thermocycler (MJ Research, Massachusetts) with a total RNA input of 50 ng according to the standard protocol of the manufacturer (Quanta Biosciences, Maryland).…”

The Novel Two-Component Regulatory System BfiSR Regulates Biofilm Development by Controlling the Small RNArsmZthrough CafA

“…Inactivation of rnr has been found to result in elevated levels and/or half-lives of over 100 transcripts in Pseudomonas putida (16). It is thus possible that rsmY degradation may be dependent on exoribonuclease RNase R under biofilm growth conditions.…”