Regulation of Bacterial Ribonucleases.

Ribonucleases (RNases) are essential for almost every aspect of RNA metabolism. However, despite their important metabolic roles, RNases can also be destructive enzymes. As a consequence, cells must carefully regulate the amount, the activity, and the localization of RNases to avoid the inappropriate degradation of essential RNA molecules. In addition, bacterial cells often must adjust RNase levels as environmental situations demand, also requiring careful regulation of these critical enzymes. As the need for strict control of RNases has become more evident, multiple mechanisms for this regulation have been identified and studied, and these are described in this review. The major conclusion that emerges is that no common regulatory mechanism applies to all RNases, or even to a family of RNases; rather, a wide variety of processes have evolved that act on these enzymes, and in some cases, multiple regulatory mechanisms can even act on a single RNase.

Hfq and RNase R Mediate rRNA Processing and Degradation in a Novel RNA Quality Control Process.

RNA quality control pathways are critical for cell survival. Here, we describe a new surveillance process involved in the degradation of highly structured and stable ribosomal RNAs. The results demonstrated that the RNA chaperone Hfq and the 3'-5' exoribonuclease R mediate the elimination of detrimental rRNA fragments and are required for the correct processing of rRNA precursors. Escherichia coli cells lacking both Hfq and RNase R accumulate a high level of 16S- and 23S-derived rRNA fragments. Hfq and RNase R were also shown to participate in the maturation of 16S and 23S rRNA precursors. This correlates with the fact that in the absence of Hfq and RNase R, there are severe ribosome assembly defects and a sharp reduction in 70S ribosome levels. Hfq and RNase R may act independently or in a complex, as protein interaction studies revealed that these RNA-binding proteins can associate. This is the first demonstration that the well-conserved Hfq and RNase R proteins act on common regulatory pathways, unraveling previously unknown mechanisms of rRNA surveillance with important consequences for translation and cell survival.IMPORTANCE Quality control pathways that oversee the quality of stable RNA molecules are critical for the cell. In this work, we demonstrate, for the first time, a functional link between Hfq and RNase R in the processing and degradation of the highly structured rRNAs. These RNA-binding proteins are required for the maturation of 16S and 23S rRNAs and correct ribosome assembly. Furthermore, they participate in the degradation of rRNAs and clearance of toxic rRNA fragments from the cell. Our studies have also shown that Hfq and RNase R can form a complex. In summary, the cooperation between Hfq and RNase R in metabolic pathways of stable RNAs may represent a broader mechanism of RNA quality control, given the high conservation of these RNA-binding proteins throughout evolution.

The CK1α Activator Pyrvinium Enhances the Catalytic Efficiency (kcat/Km) of CK1α.

The serine/threonine protein kinase casein kinase 1α (CK1α) functions as a negative regulator of Wnt signaling, phosphorylating ß-catenin at serine 45 (P-S45) to initiate its eventual ubiquitin-mediated degradation. We previously showed that the repurposed, FDA-approved anthelminthic drug pyrvinium potently inhibits Wnt signaling in vitro and in vivo. Moreover, we proposed that pyrvinium's Wnt inhibitory activity was the result of its function as an activator of CK1α. An understanding of the mechanism by which pyrvinium activates CK1α is important because pyrvinium was given an orphan drug designation by the FDA to treat familial adenomatous polyposis, a precancerous condition driven by constitutive Wnt signaling. In the current study, we show that pyrvinium stimulates the phosphorylation of S45 ß-catenin, a known CK1α substrate, in a cell-based assay, and does so in a dose- and time-dependent manner. Alternative splicing of CK1α results in four forms of the protein with distinct biological properties. We evaluated these splice products and identified the CK1α splice variant, CK1αS, as the form that exhibits the most robust response to pyrvinium in cells. Kinetic studies indicate that pyrvinium also stimulates the kinase activity of purified, recombinant CK1αS in vitro, increasing its catalytic efficiency (kcat/Km) toward substrates. These studies provide strong and clear mechanistic evidence that pyrvinium enhances CK1α kinase activity.

A novel mechanism of ribonuclease regulation: GcvB and Hfq stabilize the mRNA that encodes RNase BN/Z during exponential phase.

RNase BN, the Escherichia coli RNase Z family member, plays a limited role in tRNA metabolism, in contrast to most other organisms. However, RNase BN does act on 6S RNA, the global transcription regulator, degrading it in exponential-phase cells and maintaining it at low levels during this phase of growth. RNase BN levels decrease in stationary-phase cells, leading to elevation of 6S RNA and subsequent regulation of RNA polymerase. These findings were the first indication that RNase BN itself is growth phase-regulated. Here, we analyze the mechanism of this regulation of RNase BN. We find that RNase BN decreases in stationary phase because its mRNA becomes unstable, due primarily to its degradation by RNase E. However, in exponential-phase cells rbn mRNA is stabilized due to binding by the sRNA, GcvB, and the protein, Hfq, which reduce cleavage by RNase E. Because the amount of GcvB decreases in stationary phase, rbn mRNA is less protected and becomes increasingly unstable resulting in reduction in the amount of RNase BN. The small RNA-dependent, positive regulation of RNase BN in exponential-phase cells is the first example of this novel mechanism for RNase regulation.

Bacterial ribonucleases and their roles in RNA metabolism.

Ribonucleases (RNases) are mediators in most reactions of RNA metabolism. In recent years, there has been a surge of new information about RNases and the roles they play in cell physiology. In this review, a detailed description of bacterial RNases is presented, focusing primarily on those from Escherichia coli and Bacillus subtilis, the model Gram-negative and Gram-positive organisms, from which most of our current knowledge has been derived. Information from other organisms is also included, where relevant. In an extensive catalog of the known bacterial RNases, their structure, mechanism of action, physiological roles, genetics, and possible regulation are described. The RNase complement of E. coli and B. subtilis is compared, emphasizing the similarities, but especially the differences, between the two. Included are figures showing the three major RNA metabolic pathways in E. coli and B. subtilis and highlighting specific steps in each of the pathways catalyzed by the different RNases. This compilation of the currently available knowledge about bacterial RNases will be a useful tool for workers in the RNA field and for others interested in learning about this area.

RNase II regulates RNase PH and is essential for cell survival during starvation and stationary phase.

RNase II is the most active exoribonuclease in Escherichia coli cell extracts. Yet, its removal appears to have no deleterious effect on growing cells. Here, we show that RNase II is required for cell survival during prolonged stationary phase and upon starvation. The absence of RNase II leads to greatly increased rRNA degradation and to the accumulation of rRNA fragments, both of which lead to a decline in cell survival. The deleterious effects of RNase II removal can be completely reversed by the simultaneous absence of a second exoribonuclease, RNase PH, an enzyme known to be required to initiate ribosome degradation in starving cells. We have now found that the role of RNase II in this process is to regulate the amount of RNase PH present in starving cells, and it does so at the level of RNase PH stability. RNase PH normally decreases as much as 90% during starvation because the protein is unstable under these conditions; however, in the absence of RNase II the amount of RNase PH remains relatively unchanged. Based on these observations, we propose that in the presence of RNase II, nutrient deprivation leads to a dramatic reduction in the amount of RNase PH, thereby limiting the extent of rRNA degradation and ensuring cell survival during this stress. In the absence of RNase II, RNase PH levels remain high, leading to excessive ribosome loss and ultimately to cell death. These findings provide another example of RNase regulation in response to environmental stress.

Growth Phase-dependent Variation of RNase BN/Z Affects Small RNAs: REGULATION OF 6S RNA.

RNase BN, the RNase Z family member in E. coli, can participate in the processing of tRNA precursors. However, this function only becomes apparent when other processing enzymes are absent, raising the question of its primary physiological role. Here, we show that RNase BN itself is subject to growth phase-dependent regulation, because both rbn mRNA and RNase BN protein are at their highest levels in early exponential phase, but then decrease dramatically and are essentially absent in stationary phase. As a consequence of this variation, certain small RNAs, such as 6S RNA, remain low in exponential phase cells, and increase greatly in stationary phase. RNase BN affects 6S RNA abundance by decreasing its stability in exponential phase. RNase BN levels increase rapidly as cells exit stationary phase and are primarily responsible for the decrease in 6S RNA that accompanies this process. Purified RNase BN directly cleaves 6S RNA as shown by in vitro assays, and the 6S RNA:pRNA duplex is an even more favorable substrate of RNase BN. The exoribonuclease activity of RNase BN is unnecessary because all its action on 6S RNA is due to endonucleolytic cleavages. These data indicate that RNase BN plays an important role in determining levels of the global transcription regulator, 6S RNA, throughout the growth cycle.

Elucidation of pathways of ribosomal RNA degradation: an essential role for RNase E.

Although normally stable in growing cells, ribosomal RNAs are degraded under conditions of stress, such as starvation, and in response to misassembled or otherwise defective ribosomes in a process termed RNA quality control. Previously, our laboratory found that large fragments of 16S and 23S rRNA accumulate in strains lacking the processive exoribonucleases RNase II, RNase R, and PNPase, implicating these enzymes in the later steps of rRNA breakdown. Here, we define the pathways of rRNA degradation in the quality control process and during starvation, and show that the essential endoribonuclease, RNase E, is required to make the initial cleavages in both degradative processes. We also present evidence that explains why the exoribonuclease, RNase PH, is required to initiate the degradation of rRNA during starvation. The data presented here provide the first detailed description of rRNA degradation in bacterial cells.

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

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.

How RNase R Degrades Structured RNA: ROLE OF THE HELICASE ACTIVITY AND THE S1 DOMAIN.

RNase R, a ubiquitous 3' exoribonuclease, plays an important role in many aspects of RNA metabolism. In contrast to other exoribonucleases, RNase R can efficiently degrade highly structured RNAs, but the mechanism by which this is accomplished has remained elusive. It is known that RNase R contains an unusual, intrinsic RNA helicase activity that facilitates degradation of duplex RNA, but how it stimulates the nuclease activity has also been unclear. Here, we have made use of specifically designed substrates to compare the nuclease and helicase activities of RNase R. We have also identified and mutated several residues in the S1 RNA-binding domain that are important for interacting with duplex RNA and have measured intrinsic tryptophan fluorescence to analyze the conformational changes that occur upon binding of structured RNA. Using these approaches, we have determined the relation of the RNA helicase, ATP binding, and nuclease activities of RNase R. This information has been combined with a structural analysis of RNase R, based on its homology to RNase II, whose structure has been determined, to develop a detailed model that explains how RNase R digests structured RNA and how this differs from its action on single-stranded RNA.

Reversible acetylation on Lys501 regulates the activity of RNase II.

RNase II, a 3' to 5' processive exoribonuclease, is the major hydrolytic enzyme in Escherichia coli accounting for ∼90% of the total activity. Despite its importance, little is actually known about regulation of this enzyme. We show here that one residue, Lys501, is acetylated in RNase II. This modification, reversibly controlled by the acetyltransferase Pka, and the deacetylase CobB, affects binding of the substrate and thus decreases the catalytic activity of RNase II. As a consequence, the steady-state level of target RNAs of RNase II may be altered in the cells. We also find that under conditions of slowed growth, the acetylation level of RNase II is elevated and the activity of RNase II decreases, emphasizing the importance of this regulatory process. These findings indicate that acetylation can regulate the activity of a bacterial ribonuclease.

REP sequences: Mediators of the environmental stress response?

Repetitive Extragenic Palindromic (REP) sequences are highly conserved, structured, 35- to 40-nt elements located at ∼500 positions around the Escherichia coli chromosome. They are found in intergenic regions and are transcribed together with their upstream genes. Although their stable stem-loop structures protect messages against exoribonuclease digestion, their primary function has remained unknown. Recently, we found that about half of all REP sequences have the potential to stall ribosomes immediately upstream of the termination codon, leading to endonucleolytic cleavage of the mRNA, and induction of the trans-translation process. As a consequence, the mRNA and almost completed protein are degraded, and protein production from the affected gene is down-regulated. The process is critically dependent on the location of the REP element, with an effect only if it is within 15 nt of the termination codon. Using nrdAB as a model, we found that its down-regulation is affected by RNA helicases. Elimination of 6 helicases lowered NrdA production further, whereas overexpression of any RNA helicase partially reversed the downregulation. UV stress completely reversed down-regulation of NrdA production. Analysis of genes containing a REP sequence within 15 nt of the termination codon revealed that most, if not all, are up-regulated by environmental stress, as are RNA helicases. Based on these findings, we propose that REP-dependent downregulation serves as a mechanism to allow a rapid response to environmental stresses whereby RNA helicases partially open the REP elements enabling ribosomes to complete translation immediately increasing protein production from the affected genes.

The Helicase Activity of Ribonuclease R Is Essential for Efficient Nuclease Activity.

RNase R, which belongs to the RNB family of enzymes, is a 3' to 5' hydrolytic exoribonuclease able to digest highly structured RNA. It was previously reported that RNase R possesses an intrinsic helicase activity that is independent of its ribonuclease activity. However, the properties of this helicase activity and its relationship to the ribonuclease activity were not clear. Here, we show that helicase activity is dependent on ATP and have identified ATP-binding Walker A and Walker B motifs that are present in Escherichia coli RNase R and in 88% of mesophilic bacterial genera analyzed, but absent from thermophilic bacteria. We also show by mutational analysis that both of these motifs are required for helicase activity. Interestingly, the Walker A motif is located in the C-terminal region of RNase R, whereas the Walker B motif is in its N-terminal region implying that the two parts of the protein must come together to generate a functional ATP-binding site. Direct measurement of ATP binding confirmed that ATP binds only when double-stranded RNA is present. Detailed analysis of the helicase activity revealed that ATP hydrolysis is not required because both adenosine 5'-O-(thiotriphosphate) and adenosine 5'-(ß,γ-imino)triphosphate can stimulate helicase activity, as can other nucleoside triphosphates. Although the nuclease activity of RNase R is not needed for its helicase activity, the helicase activity is important for effective nuclease activity against a dsRNA substrate, particularly at lower temperatures and with more stable duplexes. Moreover, competition experiments and mutational analysis revealed that the helicase activity utilizes the same catalytic channel as the nuclease activity. These findings indicate that the helicase activity plays an essential role in the catalytic efficiency of RNase R.

How bacterial cells keep ribonucleases under control.

Ribonucleases (RNases) play an essential role in essentially every aspect of RNA metabolism, but they also can be destructive enzymes that need to be regulated to avoid unwanted degradation of RNA molecules. As a consequence, cells have evolved multiple strategies to protect RNAs against RNase action. They also utilize a variety of mechanisms to regulate the RNases themselves. These include post-transcriptional regulation, post-translational modification, trans-acting inhibitors, cellular localization, as well as others that are less well studied. In this review, I will briefly discuss how RNA molecules are protected and then examine in detail our current understanding of the mechanisms known to regulate individual RNases.

A role for REP sequences in regulating translation.

Repetitive extragenic palindromic (REP) sequences are highly structured elements found downstream of ∼500 genes in Escherichia coli that result in extensive stem-loop structures in their mRNAs. However, their physiological role has remained elusive. Here, we show that REP sequences can downregulate translation, but only if they are within 15 nt of a termination codon; a spacing of 16 nt has no effect, suggesting that the REP element acts to stall ribosome movement. Ribosome stalling leads to cleavage of the mRNA and induction of the trans-translation process. Using nrdAB as a model, we find that its regulation can be partially reversed by overexpression of RNA helicases and can be fully overcome upon UV stress, emphasizing the importance of this regulatory process. Since 50% of REP-associated genes have these elements within the critical 15 nt, these findings identify a regulatory mechanism with the potential to affect translation from a large number of genes.

Human DNA Exonuclease TREX1 Is Also an Exoribonuclease That Acts on Single-stranded RNA.

3' repair exonuclease 1 (TREX1) is a known DNA exonuclease involved in autoimmune disorders and the antiviral response. In this work, we show that TREX1 is also a RNA exonuclease. Purified TREX1 displays robust exoribonuclease activity that degrades single-stranded, but not double-stranded, RNA. TREX1-D200N, an Aicardi-Goutieres syndrome disease-causing mutant, is defective in degrading RNA. TREX1 activity is strongly inhibited by a stretch of pyrimidine residues as is a bacterial homolog, RNase T. Kinetic measurements indicate that the apparent Km of TREX1 for RNA is higher than that for DNA. Like RNase T, human TREX1 is active in degrading native tRNA substrates. Previously reported TREX1 crystal structures have revealed that the substrate binding sites are open enough to accommodate the extra hydroxyl group in RNA, further supporting our conclusion that TREX1 acts on RNA. These findings indicate that its RNase activity needs to be taken into account when evaluating the physiological role of TREX1.

Ribosomes regulate the stability and action of the exoribonuclease RNase R.

Ribonucleases play an important role in RNA metabolism. Yet, they are also potentially destructive enzymes whose activity must be controlled. Here we describe a novel regulatory mechanism affecting RNase R, a 3' to 5' exoribonuclease able to act on essentially all RNAs including those with extensive secondary structure. Most RNase R is sequestered on ribosomes in growing cells where it is stable and participates in trans-translation. In contrast, the free form of the enzyme, which is deleterious to cells, is extremely unstable, turning over with a half-life of 2 min. RNase R binding to ribosomes is dependent on transfer-messenger RNA (tmRNA)-SmpB, nonstop mRNA, and the modified form of ribosomal protein S12. Degradation of the free form of RNase R also requires tmRNA-SmpB, but this process is independent of ribosomes, indicating two distinct roles for tmRNA-SmpB. Inhibition of RNase R binding to ribosomes leads to slower growth and a massive increase in RNA degradation. These studies indicate a previously unknown role for ribosomes in cellular homeostasis.

How a CCA sequence protects mature tRNAs and tRNA precursors from action of the processing enzyme RNase BN/RNase Z.

In many organisms, 3' maturation of tRNAs is catalyzed by the endoribonuclease, RNase BN/RNase Z, which cleaves after the discriminator nucleotide to generate a substrate for addition of the universal CCA sequence. However, tRNAs or tRNA precursors that already contain a CCA sequence are not cleaved, thereby avoiding a futile cycle of removal and readdition of these essential residues. We show here that the adjacent C residues of the CCA sequence and an Arg residue within a highly conserved sequence motif in the channel leading to the RNase catalytic site are both required for the protective effect of the CCA sequence. When both of these determinants are present, CCA-containing RNAs in the channel are unable to move into the catalytic site; however, substitution of either of the C residues by A or U or mutation of Arg(274) to Ala allows RNA movement and catalysis to proceed. These data define a novel mechanism for how tRNAs are protected against the promiscuous action of a processing enzyme.