Helicase Activity Plays a Crucial Role for RNase R Function in Vivo and for RNA Metabolism

Abstract

RNase R is a 3 to 5 hydrolytic exoribonuclease that has the unusual ability to digest highly structured RNA. The enzyme possesses an intrinsic, ATP-dependent RNA helicase activity that is essential in vitro for efficient nuclease activity against double-stranded RNA substrates, particularly at lower temperatures, with more stable RNA duplexes, and for duplexes with short 3 overhangs. Here, we inquired whether the helicase activity was also important for RNase R function in vivo and for RNA metabolism. We find that strains containing a helicase-deficient RNase R due to mutations in its ATP-binding Walker motifs exhibit growth defects at low temperatures. Most importantly, cells also lacking polynucleotide phosphorylase (PNPase), and dependent for growth on RNase R, grow extremely poorly at 34, 37, and 42°C and do not grow at all at 31°C. Northern analysis revealed that in these cells, fragments of 16S and 23S rRNA accumulate to high levels, leading to interference with ribosome maturation and ultimately to cell death. These findings indicate that the intrinsic helicase activity of RNase R is required for its proper functioning in vivo and for effective RNA metabolism.RNase R and its homologues are processive, 3Ј to 5Ј exoribonucleases present in organisms from bacteria to higher eukaryotes (1). These enzymes play important roles in many aspects of RNA metabolism including mRNA and stable RNA turnover (2, 3) and rRNA maturation (4). In contrast to most exoribonucleases, RNase R is particularly adept at degrading highly structured RNA molecules (2, 5-7), and defining the mechanism by which it carries out this process is highly important (8, 9). Escherichia coli RNase R contains an intrinsic RNA helicase activity (10), and recent work from our laboratory characterized this helicase and defined its relation to overall nuclease activity (8, 9). In the E. coli protein, we also identified Walker A and Walker B motifs that are responsible for ATP binding and consequent RNA helicase activity, and found that they are conserved in most mesophilic bacterial genera, but are absent from thermophilic bacteria (8), suggesting that the helicase is important for RNase R function. This conclusion was reinforced by biochemical analyses showing that the helicase activity is essential for efficient nuclease activity in vitro, particularly at lower temperatures, with duplexes containing short 3Ј overhangs, and with more stable RNA duplexes (8, 9). Whether the helicase activity is also important for nuclease activity in vivo has been unclear.It is known that RNase R can complement the essential function of CsdA, a DEAD-box RNA helicase, during cold shock (11), implying that the helicase activity of RNase R can function in vivo. Moreover, this complementation activity persists even when the nuclease activity of RNase R has been eliminated by mutation. However, all of these studies were carried out under conditions in which RNase R was overexpressed, raising the question of whether the helicase ever functions physiologically in vivo indep...

Bacterial ribonucleases and their roles in RNA metabolism

“…The helicase activity utilizes the same catalytic channel as the nuclease activity, and is an intrinsic component of the enzyme’s ability to digest structured RNAs since mutations that interfere with ATP binding prevent digestion of structured RNA, but have no effect on the digestion of single-stranded RNA molecules (Hossain et al 2016). Escherichia coli strains in which the helicase activity of RNase R has been eliminated by mutation of the Walker motifs exhibit growth defects at low temperatures (Hossain and Deutscher 2016). Moreover, cells also lacking PNPase and dependent on RNase R for growth, do not grow at 31 °C, and grow extremely poorly at 34, 37, and 42 °C.…”

“…Although very little work has been carried out on expression of the rnr gene (Cairrao et al 2003; Cairrao and Arraiano 2006), it appears to be part of an operon that contains the nsrR gene (coding for a NO-dependent transcriptional repressor) upstream, and rlmB (encoding a 23S rRNA methyltransferase) and yjfI downstream. Mutant strains lacking RNase R grow essentially normally at temperatures between 31 and 42 °C, but display slowed growth at temperatures of 25 °C and below (Cairrao et al 2003; Hossain and Deutscher 2016). In contrast, a mutant strain lacking both RNase R and PNPase does not grow (Cheng and Deutscher 2003), suggesting overlap in an essential cellular function.…”

“…Although E. coli cells devoid of PNPase retain viability, their growth rate slows (McMurry and Levy 1987), and this becomes much more pronounced as the growth temperature decreases (Hossain and Deutscher 2016). PNPase is an important participant in many RNA metabolic reactions, and its absence affects multiple cellular processes (Briani et al 2016).…”

Role of ribosome assembly in Escherichia coli ribosomal RNA degradation

“…High levels of rRNA degradation fragments have been observed before in pnp ts Δ rnr strains ( 24 ), but those studies left open the possibility that they could have arisen due to the treatment applied to inactivate PNPase, a condition that leads to growth cessation. Similarly, rRNA fragment accumulation has also been observed in strains that lack PNPase and contain mutations in RNase R that inactivate its helicase activity, but these strains exhibit a four-fold increase in cell doubling time ( 38 ). In contrast, another study found undetectable levels of rRNA degradation over several hours of cell growth ( 31 ).…”

RNA helicases in RNA decay

“…The helicase activity does not require the hydrolytic exoribonuclease activity; however, the nuclease activity on double-stranded RNA requires the helicase activity [ 43 ]. Importantly, the helicase activity is required in vivo especially at low temperature [ 44 ].…”