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A key pathway for mRNA degradation in bacterial cells begins with conversion of the initial 5′-terminal triphosphate to a monophosphate, a modification that renders transcripts more vulnerable to attack by ribonucleases whose affinity for monophosphorylated 5′ ends potentiates their catalytic efficacy. In Escherichia coli, the only proteins known to be important for controlling degradation via this pathway are the RNA pyrophosphohydrolase RppH, its heteromeric partner DapF, and the 5′-monophosphate-assisted endonucleases RNase E and RNase G. We have now identified the metabolic enzyme cytidylate kinase as another protein that affects rates of 5′-end-dependent mRNA degradation in E. coli. It does so by utilizing two distinct mechanisms to influence the 5′-terminal phosphorylation state of RNA, each dependent on the catalytic activity of cytidylate kinase and not its mere presence in cells. First, this enzyme acts in conjunction with DapF to stimulate the conversion of 5′ triphosphates to monophosphates by RppH. In addition, it suppresses the direct synthesis of monophosphorylated transcripts that begin with cytidine by reducing the cellular concentration of cytidine monophosphate, thereby disfavoring the 5′-terminal incorporation of this nucleotide by RNA polymerase during transcription initiation. Together, these findings suggest dual signaling pathways by which nucleotide metabolism can impact mRNA degradation in bacteria.

In countless bacterial species, the lifetimes of most mRNAs are controlled by the regulatory endonuclease RNase E, which preferentially degrades RNAs bearing a 5′ monophosphate and locates cleavage sites within them by scanning linearly from the 5′ terminus along single-stranded regions. Consequently, its rate of cleavage at distal sites is governed by any obstacles that it may encounter along the way, such as bound proteins or ribosomes or base pairing that is coaxial with the path traversed by this enzyme. Here, we report that the protection afforded by such obstacles is dependent on the size and persistence of the structural discontinuities they create, whereas the molecular composition of obstacles to scanning is of comparatively little consequence. Over a broad range of sizes, incrementally larger discontinuities are incrementally more protective, with corresponding effects on mRNA stability. The graded impact of such obstacles suggests possible explanations for why their effect on scanning is not an all-or-none phenomenon dependent simply on whether the size of the resulting discontinuity exceeds the step length of RNase E.

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np(4)Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np(4)) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np(4)Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np(4)-capped in E. coli, clear evidence for Np(4) cap acquisition by Np(4)N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np(4)As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np(4)Ns function in bacteria as precursors to Np(4) caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

Significance Dinucleoside tetraphosphate alarmones function in bacteria as precursors to 5′-terminal nucleoside tetraphosphate (Np 4 ) caps, becoming incorporated at high levels into RNA during stress and thereby influencing transcript lifetimes. However, little is known about how these noncanonical caps are removed as a prelude to RNA degradation. Here, we report that the RNA pyrophosphohydrolase RppH assumes a leading role in decapping those transcripts under conditions of disulfide stress and that it recognizes Np 4 -capped 5′ ends by an unexpected mechanism, generating a triphosphorylated RNA intermediate that must undergo further deprotection by RppH to trigger degradation. These findings help to explain the uneven distribution of Np 4 caps on bacterial transcripts and have important implications for how gene expression is reprogrammed in response to stress.

Dinucleoside tetraphosphates, often described as alarmones because their cellular concentration increases in response to stress, have recently been shown to function in bacteria as precursors to nucleoside tetraphosphate (Np

SummaryA distinguishing feature of Trichuris nematodes is that these parasitic worms reproduce within the digestive tracts of humans and other mammalian hosts shedding thousands of eggs daily, facilitating their sustained presence in the environment and hampering eradication efforts. Although this aspect of the lifecycle places Trichuris in a microbiota-rich environment, metabolic byproducts of bacteria that facilitate the reproductive development of parasites are unknown. Here, we employ a pipeline using the well-characterized free-living nematode C. elegans to identify microbial factors with conserved roles in the reproduction of nematodes. A screen for E. coli mutants that impair C. elegans fertility identified genes in fatty acid biosynthesis and ethanolamine utilization pathways, including fabH and eutN. Trichuris muris eggs displayed defective hatching in the presence of E. coli deficient in fabH or eutN due to reduction in arginine or elevated levels of aldehydes, respectively. Remarkably, T. muris reared in gnotobiotic mice colonized with these E. coli mutants displayed profound abnormalities including morphological defects and a failure to lay viable eggs. These findings indicate that microbial byproducts mediate evolutionarily conserved transkingdom interactions that impact the reproductive fitness of distantly-related nematodes.

Although riboswitches have long been known to regulate translation initiation and transcription termination, a growing body of evidence indicates that they can also control bacterial RNA lifetimes by acting directly to hasten or impede RNA degradation. Ligand binding to the aptamer domain of a riboswitch can accelerate RNA decay by triggering a conformational change that exposes sites to endonucleolytic cleavage or by catalyzing the self-cleavage of a prefolded ribozyme. Alternatively, the conformational change induced by ligand binding can protect RNA from degradation by blocking access to an RNA terminus or internal region that would otherwise be susceptible to attack by an exonuclease or an endonuclease. Such changes in RNA longevity often accompany a parallel effect of the same riboswitch on translation or transcription. Consequently, a single riboswitch aptamer may govern the function of multiple effector elements (expression platforms) that are co-resident within a transcript and act independently of one another.

Riboswitches are thought generally to function by modulating transcription elongation or translation initiation. In rare instances, ligand binding to a riboswitch has been found to alter the rate of RNA degradation by directly stimulating or inhibiting nearby cleavage. Here we show that guanidine-induced pseudoknot formation by the aptamer domain of a guanidine III riboswitch from Legionella pneumophila has a very different effect, stabilizing mRNA by protecting distal cleavage sites en masse from ribonuclease attack. It does so by creating a coaxially base-paired obstacle that impedes scanning from a monophosphorylated 5′ end to those sites by the regulatory endonuclease RNase E. Ligand binding by other riboswitch aptamers peripheral to the path traveled by RNase E does not inhibit distal cleavage. These findings reveal that a riboswitch aptamer can function independently of any overlapping expression platform to regulate gene expression by acting directly to prolong mRNA longevity in response to ligand binding.

Vitally important for controlling gene expression in eukaryotes and prokaryotes, the deprotection of mRNA 5′ termini is governed by enzymes whose activity is modulated by interactions with ancillary factors. In Escherichia coli, 5′-end-dependent mRNA degradation begins with the generation of monophosphorylated 5′ termini by the RNA pyrophosphohydrolase RppH, which can be stimulated by DapF, a diaminopimelate epimerase involved in amino acid and cell wall biosynthesis. We have determined crystal structures of RppH–DapF complexes and measured rates of RNA deprotection. These studies show that DapF potentiates RppH activity in two ways, depending on the nature of the substrate. Its stimulatory effect on the reactivity of diphosphorylated RNAs, the predominant natural substrates of RppH, requires a substrate long enough to reach DapF in the complex, while the enhanced reactivity of triphosphorylated RNAs appears to involve DapF-induced changes in RppH itself and likewise increases with substrate length. This study provides a basis for understanding the intricate relationship between cellular metabolism and mRNA decay and reveals striking parallels with the stimulation of decapping activity in eukaryotes.

RNase E and RNase G are homologous endonucleases that play important roles in RNA processing and decay in Escherichia coli and related bacterial species. Rapid mRNA degradation is facilitated by the preference of both enzymes for decay intermediates whose 5′ end is monophosphorylated. In this report we identify key characteristics of RNA that influence the rate of 5′-monophosphate-assisted cleavage by these two ribonucleases. In vitro, both require at least two and prefer three or more unpaired 5′-terminal nucleotides for such cleavage; however, RNase G is impeded more than RNase E when fewer than four unpaired nucleotides are present at the 5′ end. Each can tolerate any unpaired nucleotide (A, G, C, or U) at either of the first two positions, with only modest biases. The optimal spacing between the 5′ end and the scissile phosphate appears to be eight nucleotides for RNase E but only six for RNase G. 5′-Monophosphate-assisted cleavage also occurs, albeit more slowly, when that spacing is greater or at most one nucleotide shorter than the optimum, but there is no simple inverse relationship between increased spacing and the rate of cleavage. These properties are also manifested during 5′-end-dependent mRNA degradation in E. coli.

The function and fate of cellular RNAs are often governed by the phosphorylation state at the 5′ end or the identity of whatever cap may be present there. Here we describe methods for examining these important 5′-terminal features on any cellular or synthetic RNA of interest that can be detected by Northern blotting. One such method, PABLO, is a splinted ligation assay that makes it possible to accurately quantify the percentage of 5′ ends that are monophosphorylated. Another, PACO, is a capping assay that reveals the percentage of 5′ ends that are diphosphorylated. A third, boronate gel electrophoresis in conjunction with deoxyribozyme-mediated cleavage, enables different types of caps (e.g., m(7)Gppp caps versus NAD caps) to be distinguished from one another and the percentage of each to be determined. After completing all three tests, the percentage of 5′ ends that are triphosphorylated can be deduced by process of elimination. Together, this battery of assays allows the 5′ terminus of an RNA to be profiled in unprecedented detail.

Deprotection of the 5' end appears to be a universal mechanism for triggering the degradation of mRNA in bacteria and eukaryotes. In Escherichia coli, for example, converting the 5' triphosphate of primary transcripts to a monophosphate accelerates cleavage at internal sites by the endonuclease RNase E. Previous studies have shown that the RNA pyrophosphohydrolase RppH catalyzes this transformation in vitro and generates monophosphorylated decay intermediates in vivo. Recently, we reported that purified E. coli RppH unexpectedly reacts faster with diphosphorylated than with triphosphorylated substrates. By using a novel assay, it was also determined that diphosphorylated mRNA decay intermediates are abundant in wild-type E. coli and that their fractional level increases to almost 100% for representative mRNAs in mutant cells lacking RppH. These findings indicate that the conversion of triphosphorylated to monophosphorylated RNA in E. coli is a stepwise process involving sequential phosphate removal and the transient formation of a diphosphorylated intermediate. The latter RNA phosphorylation state, which was previously unknown in bacteria, now appears to define the preferred biological substrates of E. coli RppH. The enzyme responsible for generating it remains to be identified.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that assembles a type III secretion system (T3SS) on its surface. The last portion of the T3SS, called the 'translocon', is composed of a filament and a pore complex that is inserted into the membrane of intestinal epithelial cells. The genes encoding the translocon (espADB) are part of the LEE4 operon. Their expression is regulated by a complex post-transcriptional mechanism that involves the processing of LEE4 mRNA by the essential endoribonuclease RNase E. Here, we report the construction of an EHEC strain (TEA028-rne) in which RNase E can be induced by adding IPTG to the culture medium. EHEC cells deficient in RNase E displayed an abnormal morphology and slower growth, in agreement with published observations in E. coli K-12. Under those conditions, EspA and EspB were produced at higher concentrations, and protein secretion still occurred. These results indicate that RNase E negatively regulates translocon protein synthesis and demonstrate the utility of E. coli strain TEA028-rne as a tool for investigating the influence of this ribonuclease on EHEC gene expression in vitro.

In metazoans, cleavage by the endoribonuclease SMG6 is often the first degradative event in non-sense-mediated mRNA decay (NMD). However, the exact sites of SMG6 cleavage have yet to be determined for any endogenous targets, and most evidence as to the identity of SMG6 substrates is indirect. Here, we use Parallel Analysis of RNA Ends to specifically identify the 5' termini of decay intermediates whose production is dependent on SMG6 and the universal NMD factor UPF1. In this manner, the SMG6 cleavage sites in hundreds of endogenous NMD targets in human cells have been mapped at high resolution. In addition, a preferred sequence motif spanning most SMG6 cleavage sites has been discovered and validated by mutational analysis. For many SMG6 substrates, depletion of SMG6 resulted in the accumulation of decapped transcripts, an effect indicative of competition between SMG6-dependent and SMG6-independent NMD pathways. These findings provide key insights into the mechanisms by which mRNAs targeted by NMD are degraded.

On June 4-8, 2013, the 3rd Conference on Regulation with RNA in Bacteria took place in Wurzburg, Germany. Following two earlier meetings in Berlin and San Juan, this conference has established itself as the primary bi-annual meeting for everyone interested in RNA-based regulations in prokaryotes. The 2013 meeting was organized by Joel Belasco, Susan Gottesman, Franz Narberhaus, and Jorg Vogel. Close to 300 participants from more than 27 countries in Europe, North America, and Asia enjoyed four days of talks and posters on many experimental and biocomputational aspects of prokaryotic RNA biology.

Summary Present in all realms of life, dinucleoside tetraphosphates (Np 4 Ns) are generally considered signaling molecules. However, only a single pathway for Np 4 N signaling has been delineated in eukaryotes, and no receptor that mediates the influence of Np 4 Ns has ever been identified in bacteria. Here we show that, under disulfide stress conditions that elevate cellular Np 4 N concentrations, diverse Escherichia coli mRNAs and sRNAs acquire a cognate Np 4 cap. Purified E. coli RNA polymerase and lysyl-tRNA synthetase are both capable of adding such 5′ caps. Cap removal by either of two pyrophosphatases, ApaH or RppH, triggers rapid RNA degradation in E. coli . ApaH, the predominant decapping enzyme, functions as both a sensor and an effector of disulfide stress, which inactivates it. These findings suggest that the physiological changes attributed to elevated Np 4 N concentrations in bacteria may result from widespread Np 4 capping, leading to altered RNA stability and consequent changes in gene expression.

The diversity of mRNA lifetimes in bacterial cells is difficult to reconcile with the relaxed cleavage site specificity of RNase E, the endonuclease most important for governing mRNA degradation. This enzyme has generally been thought to locate cleavage sites by searching freely in three dimensions. However, our results now show that its access to such sites in 5'-monophosphorylated RNA is hindered by obstacles-such as bound proteins or ribosomes or coaxial small RNA (sRNA) base pairing-that disrupt the path from the 5' end to those sites and prolong mRNA lifetimes. These findings suggest that RNase E searches for cleavage sites by scanning linearly from the 5'-terminal monophosphate along single-stranded regions of RNA and that its progress is impeded by structural discontinuities encountered along the way. This discovery has major implications for gene regulation in bacteria and suggests a general mechanism by which other prokaryotic and eukaryotic regulatory proteins can be controlled.

Bacterial RNA degradation often begins with conversion of the 5′-terminal triphosphate to a monophosphate, creating a better substrate for subsequent ribonuclease digestion. For example, in Bacillus subtilis and related organisms, removal of the gamma and beta phosphates of primary transcripts by the RNA pyrophosphohydrolase RppH triggers rapid 5′-exonucleolytic degradation by RNase J. However, the basis for the selective targeting of a subset of cellular RNAs by this pathway has remained largely unknown. Here we report that purified B. subtilis RppH requires at least two unpaired nucleotides at the 5′ end of its RNA substrates and prefers three or more. The second of these 5′-terminal nucleotides must be G, whereas a less strict preference for a purine is evident at the third position, and A is slightly favored over G at the first position. The same sequence requirements are observed for RppH-dependent mRNA degradation in B. subtilis cells. By contrast, a parallel pathway for 5′-end–dependent RNA degradation in that species appears to involve an alternative phosphate-removing enzyme that is relatively insensitive to sequence variation at the first three positions.

Many Escherichia coli mRNAs are degraded by a 5′-end-dependent mechanism in which RppH-catalyzed conversion of the 5′-terminal triphosphate to a monophosphate triggers rapid endonucleolytic cleavage by RNase E. However, little is understood about what governs the decay rates of these transcripts. We investigated the decay of three such messages— rpsT P1, yfcZ , and ydfG— to characterize the rate-determining step in their degradation. The steady-state ratio of monophosphorylated to triphosphorylated rpsT P1 and yfcZ mRNA indicates that their decay rate is limited by cleavage of the monophosphorylated intermediate, making RNase E critical for their rapid turnover. Conversely, the decay rate of ydfG is limited by generation of the monophosphorylated intermediate; therefore, either RNase E or its less abundant paralog RNase G is sufficient for rapid ydfG degradation. Although all three transcripts are stabilized when RppH is absent, overproducing RppH does not accelerate their decay, nor does RppH overproduction appear to influence the longevity of most other messages that it targets. The failure of excess RppH to hasten rpsT P1 and yfcZ degradation despite increasing the percentage of each that is monophosphorylated is consistent with the observation that pyrophosphate removal is not the rate-limiting step in their decay. In contrast, neither the ydfG decay rate nor the fraction of ydfG transcripts that are monophosphorylated increases when the cellular concentration of RppH is raised, suggesting that, for some RppH targets, the rate of formation of the monophosphorylated intermediate is limited by an ancillary factor or by a step that precedes pyrophosphate removal.

Enterohaemorrhagic Escherichia coli harbours a pathogenicity island encoding a type 3 secretion system used to translocate effector proteins into the cytosol of intestinal epithelial cells and subvert their function. The structural proteins of the translocon are encoded in a major espADB mRNA processed from a precursor. The translocon mRNA should be highly susceptible to RNase E cleavage because of its AU-rich leader region and monophosphorylated 5'-terminus, yet it manages to avoid rapid degradation. Here, we report that the espADB leader region contains a strong Shine-Dalgarno element (SD2) and a translatable mini-ORF of six codons. Disruption of SD2 so as to weaken ribosome binding significantly reduces the concentration and stability of esp mRNA, whereas codon substitutions that impair translation of the mini-ORF have no such effect. These findings suggest that occupancy of SD2 by ribosomes, but not mini-ORF translation, helps to protect espADB mRNA from degradation, likely by hindering RNase E access to the AU-rich leader region.

Summary In Escherichia coli, the endonuclease RNase E can access internal cleavage sites in mRNA either directly or by a 5′ end-dependent mechanism in which cleavage is facilitated by prior RppH-catalysed conversion of the 5′-terminal triphosphate to a monophosphate, to which RNase E can bind. The characteristics of transcripts that determine which of these two pathways is primarily responsible for their decay are poorly understood. Here we report the influence of ribosome binding and translocation on each pathway, using yeiP and trxB as model transcripts. Ribosome binding to the translation initiation site impedes degradation by both mechanisms. However, because the effect on the rate of 5′ end-independent decay is greater, poor ribosome binding favours degradation by that pathway. Arresting translation elongation with chloramphenicol quickly inhibits RNase E cleavage downstream of the initiation codon but has little or no immediate effect on cleavage upstream of the ribosome binding site. RNase E binding to a monophosphorylated 5′ end appears to increase the likelihood of cleavage at sites within the 5′ untranslated region. These findings indicate that ribosome binding and translocation can have a major impact on 5′ end-dependent mRNA degradation in E. coli and suggest a possible sequence of events that follow pyrophosphate removal.

In this issue of Molecular Cell, Chao et al. (2017) investigate the important role of the low-specificity endonuclease RNase E in shaping the transcriptome of a bacterial pathogen by functioning as both a degradative enzyme and an RNA maturase.

In Escherichia coli, RNA degradation often begins with conversion of the 5'-terminal triphosphate to a monophosphate, creating a better substrate for internal cleavage by RNase E. Remarkably, no homolog of this key endonuclease is present in many bacterial species, such as Bacillus subtilis and various pathogens. Here, we report that the degradation of primary transcripts in B. subtilis can nevertheless be triggered by an analogous process to generate a short-lived, monophosphorylated intermediate. Like its E. coli counterpart, the B. subtilis RNA pyrophosphohydrolase that catalyzes this event is a Nudix protein that prefers unpaired 5' ends. However, in B. subtilis, this modification exposes transcripts to rapid 5' exonucleolytic degradation by RNase J, which is absent in E. coli but present in most bacteria lacking RNase E. This pathway, which closely resembles the mechanism by which deadenylated mRNA is degraded in eukaryotic cells, explains the stabilizing influence of 5'-terminal stem-loops in such bacteria.

Regulated transport and local translation of mRNA in neurons are critical for modulating synaptic strength, maintaining proper neural circuitry, and establishing long term memory. Neuronal RNA granules are ribonucleoprotein particles that serve to transport mRNA along microtubules and control local protein synthesis in response to synaptic activity. Studies suggest that neuronal RNA granules share similar structures and functions with somatic P-bodies. We recently reported that the Huntington disease protein huntingtin (Htt) associates with Argonaute (Ago) and localizes to cytoplasmic P-bodies, which serve as sites of mRNA storage, degradation, and small RNA-mediated gene silencing. Here we report that wild-type Htt associates with Ago2 and components of neuronal granules and co-traffics with mRNA in dendrites. Htt was found to co-localize with RNA containing the 3'-untranslated region sequence of known dendritically targeted mRNAs. Knockdown of Htt in neurons caused altered localization of mRNA. When tethered to a reporter construct, Htt down-regulated reporter gene expression in a manner dependent on Ago2, suggesting that Htt may function to repress translation of mRNAs during transport in neuronal granules.

MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of “off-target” mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal.

Summary Until recently, the mechanism of mRNA decay in bacteria was thought to be different from that of eukaryotes. This paradigm changed with the discovery that RppH (ORF176/NudH/YgdP), an Escherichia coli enzyme that belongs to the Nudix superfamily, is an RNA pyrophosphohydrolase that initiates mRNA decay by cleaving pyrophosphate from the 5′-triphosphate. Here we report the 1.9 A resolution structure of the Nudix hydrolase BdRppH from Bdellovibrio bacteriovorus , a bacterium that feeds on other Gram-negative bacteria. Based on the structure of the enzyme alone and in complex with GTP-Mg 2+ , we propose a mode of RNA binding similar to that of the nuclear decapping enzyme from Xenopus laevis , X29. In additional experiments, we show that BdRppH can indeed function in vitro and in vivo as an RNA pyrophosphohydrolase. These findings set the basis for the identification of possible decapping enzymes in other bacteria.

SummaryIn animals, both siRNAs and miRNAs are thought to diminish protein synthesis from transcripts that are perfectly complementary by directing endonucleolytic cleavage where they anneal, thereby triggering rapid degradation of the entire message [1–4]. By contrast, partially complementary messages are downregulated by a combination of translational repression and accelerated decay caused by rapid poly(A) tail removal [3, 5–12]. Here we present evidence that translational repression can also make a substantial contribution to the downregulation of fully complementary messages by RNA interference. Unlike mRNA destabilization, this inhibitory effect on translation is greater for perfectly complementary elements located in the 3′ untranslated region rather than in the protein-coding region. In addition to known disparities in their endonucleolytic activity [13, 14], the four Ago proteins with which siRNAs associate in humans differ significantly in their capacity to direct translational repression. As a result, the relative effect of siRNA on targets that are fully versus partially complementary is influenced by the comparative abundance of the three nonnucleolytic Ago proteins, causing this on-target/off-target ratio to vary in a cell-type-dependent manner because of the dissimilar tissue distribution of these proteins. These findings have important implications for the efficacy and specificity of RNA interference.

The downregulation of gene expression by miRNAs and siRNAs is a complex process involving both translational repression and accelerated mRNA turnover, each of which appears to occur by multiple mechanisms. Moreover, under certain conditions, miRNAs are also capable of activating translation. A variety of cellular proteins have been implicated in these regulatory mechanisms, yet their exact roles remain largely unresolved.

The common belief that endonucleolytic cleavage is the initial, rate-determining step of mRNA decay in Escherichia coli fails to explain the influence of 5’ termini on the half-lives of primary transcripts. We have re-examined the initial events of RNA degradation in that organism by devising an assay to probe the 5’ phosphorylation state of RNA and by employing a self-cleaving hammerhead ribozyme to investigate the degradative consequences of an unphosphorylated 5’ end. These studies have identified a previously unrecognized prior step in decay that triggers subsequent internal cleavage by the endonuclease RNase E and thereby governs RNA longevity: the rate-determining conversion of a triphosphorylated to a monophosphorylated 5’ terminus. Our findings redefine the role of RNase E in RNA degradation and explain how unpaired 5’-terminal nucleotides can facilitate access to internal cleavage sites within primary transcripts. Moreover, these results reveal a striking parallel between the mechanisms of mRNA decay in prokaryotic and eukaryotic organisms.

Although widely assumed to bear a 5′-terminal triphosphate or monophosphate, recent evidence suggests that the 5′ end of bacterial RNA can sometimes bear a modification reminiscent of a eukaryotic cap. A new study has now identified Escherichia coli RNAs that begin with a noncanonical cap resembling the redox cofactor nicotinamide adenine dinucleotide (NAD), as well as a cellular enzyme that can remove it. The biological function of such caps remains to be determined.

Background: In bacteria, RppH triggers mRNA degradation by converting the 5′-terminal triphosphate to a monophosphate. Results: The substrate specificity of E. coli RppH has been determined, along with residues important for this property and species in which they are conserved. Conclusion: The specificity of RppH is species-dependent. Significance: These findings help to explain the selective influence of RppH on rates of mRNA decay. Bacterial RNA degradation often begins with conversion of the 5′-terminal triphosphate to a monophosphate by the RNA pyrophosphohydrolase RppH, an event that triggers rapid ribonucleolytic attack. Besides its role as the master regulator of 5′-end-dependent mRNA decay, RppH is important for the ability of pathogenic bacteria to invade host cells, yet little is known about how it chooses its targets. Here, we show that Escherichia coli RppH (EcRppH) requires at least two unpaired nucleotides at the RNA 5′ end and prefers three or more such nucleotides. It can tolerate any nucleotide at the first three positions but has a modest preference for A at the 5′ terminus and either a G or A at the second position. Mutational analysis has identified EcRppH residues crucial for substrate recognition or catalysis. The promiscuity of EcRppH differentiates it from its Bacillus subtilis counterpart, which has a strict RNA sequence requirement. EcRppH orthologs likely to share its relaxed sequence specificity are widespread in all classes of Proteobacteria, except Deltaproteobacteria, and in flowering plants. By contrast, the phylogenetic range of recognizable B. subtilis RppH orthologs appears to be restricted to the order Bacillales. These findings help to explain the selective influence of RppH on bacterial mRNA decay and show that RppH-dependent degradation has diversified significantly during the course of evolution.

The lifetimes of bacterial mRNAs are strongly affected by their association with ribosomes. Events occurring at any stage during translation, including ribosome binding, polypeptide elongation, or translation termination, can influence the susceptibility of mRNA to ribonuclease attack. Ribosomes usually act as protective barriers that impede mRNA cleavage, but in some instances they can instead trigger the decay of the mRNA to which they are bound or send a signal that leads to widespread mRNA destabilization within a cell. The influence of translation on mRNA decay provides a quality-control mechanism for minimizing the use of poorly or improperly translated mRNAs as templates for the production of abnormal proteins that might be toxic to bacteria. Messenger RNA degradation plays a key role in controlling gene expression in all organisms. This is particularly true in bacteria, where mRNA decay rates can differ by as much as two orders of magnitude, with half-lives ranging from a fraction of a minute to more than an hour. The lifetimes of individual mRNAs are influenced by several factors, among the most important being their association with translating ribosomes. Recent discoveries have led to a better appreciation of the variety of ways in which ribosomes can impede or accelerate bacterial mRNA degradation and the regulatory significance of their ability to do so. Typical mechanism of mRNA decay in bacteria

RNase G is a homologue of the essential Escherichia coli ribonuclease RNase E. Whereas RNase E plays a key role in the degradation of mRNA and the processing of tRNA and rRNA in E. coli, the biological functions of RNase G appear more limited. We report here that this difference in function is not merely a consequence of the significantly lower cellular concentration of RNase G, but also reflects differences in the intrinsic properties of these ribonucleases, as overproducing wild-type RNase G at a level up to 20 times the usual cellular concentration of RNase E cannot normally compensate for the absence of RNase E in E. coli. Instead, RNase G can sustain significant growth of RNase E-deficient E. coli cells only when it bears an unnatural extension at its amino terminus (e.g. MRKGINM) or carboxyl terminus (e.g. GHHHHHH). These extensions presumably enable RNase G to cleave critically important cellular RNAs whose efficient processing or degradation ordinarily requires RNase E. That extending the amino terminus of RNase G restores growth to E. coli cells lacking RNase E without detectably improving tRNA processing suggests that RNase E is not essential for tRNA production and is required for cell growth because it plays an indispensable role in the maturation or decay of essential E. coli RNAs other than tRNA.

Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modelled the three-dimensional structure of an essential amino-terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA-binding domains. Each of the five surface-exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe-57, Phe-67 and Lys-112 [corrected], is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys-37 and Tyr-60, is entirely dispensable for catalytic activity in vitro. The side-chains of two residues previously identified as sites of temperature-sensitive mutations lie buried directly beneath the surface region defined by Phe-57, Phe-67 and Lys-112 [corrected], which probably enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. In contrast to the S1 domain, an arginine-rich RNA-binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth or ribonuclease activity.

RNase E is an important regulatory enzyme that governs the principal pathway for mRNA degradation in Escherichia coli. This endonuclease controls its own synthesis via a feedback mechanism in which the longevity of rne (RNase E) mRNA is modulated by a cis-acting sensory element that responds to changes in cellular RNase E activity. Previous research has shown that this element is an RNA stem-loop (hp2) within the 5'-untranslated region of the rne transcript. Here we report studies involving mutational analysis and phylogenetic comparison that have identified the features of rne hp2 important for its function. These comprise an internal loop flanked on one side by a 2-bp stem and a hairpin loop and on the other side by a longer stem whose sequence is inconsequential. A search of bacterial genome sequences suggests that regulation by an hp2-like element may be a unique evolutionary adaptation of the rne transcript that is not shared by other mRNAs.

Summary The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate-determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10–20% of normal and that the feedback mecha-nism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the in-crease in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein.

The labile SAUR transcripts from higher plants contain a conserved DST sequence in their 3′-untranslated regions. Two copies of a DST sequence from soybean are sufficient to destabilize reporter transcripts in cultured tobacco cells whereas variants bearing mutations in the conserved ATAGAT or GTA regions are inactive. To investigate the potential for conserved recognition components in mammalian and plant cells, we examined the function of this instability determinant in mouse NIH3T3 fibroblasts and tobacco BY2 cells. In fibroblasts, a tetrameric DST element from soybean accelerated deadenylation and decay of a reporter transcript. However, a version mutated in the ATAGAT region was equally effective in this regard, and a tetrameric DST element from Arabidopsis was inactive. In contrast, the soybean DST element was more active as an mRNA instability element than the mutant version and the Arabidopsiselement, when tested as tetramers in tobacco cells. Hence, the plant DST element is not recognized in animal cells with the same sequence requirements as in plant cells. Therefore, its mode of recognition appears to be plant-specific.

Summary RNA modifications that once escaped detection are now thought to be pivotal for governing RNA lifetimes in both prokaryotes and eukaryotes. For example, converting the 5′-terminal triphosphate of bacterial transcripts to a monophosphate triggers 5′ end-dependent degradation by RNase E. However, the existence of diphosphorylated RNA in bacteria has never been reported, and no biological role for such a modification has ever been proposed. By using a novel assay, we show here for representative Escherichia coli mRNAs that ∼35%–50% of each transcript is diphosphorylated. The remainder is primarily monophosphorylated, with surprisingly little triphosphorylated RNA evident. Furthermore, diphosphorylated RNA is the preferred substrate of the RNA pyrophosphohydrolase RppH, whose biological function was previously assumed to be pyrophosphate removal from triphosphorylated transcripts. We conclude that triphosphate-to-monophosphate conversion to induce 5′ end-dependent RNA degradation is a two-step process in E. coli involving γ-phosphate removal by an unidentified enzyme to enable subsequent β-phosphate removal by RppH.

The involvement of the recently characterized 5′ exonuclease activity of RNase J1 and endonuclease activity of RNase Y in the turnover of Δ ermC mRNA in Bacillus subtilis was investigated. Evidence is presented that both of these activities determine the half-life of Δ ermC mRNA.

mRNA degradation is an important mechanism for controlling gene expression in bacterial cells. This process involves the orderly action of a battery of cellular endonucleases and exonucleases, some universal and others present only in certain species. These ribonucleases function with the assistance of ancillary enzymes that covalently modify the 5′ or 3′ end of RNA or unwind base-paired regions. Triggered by initiating events at either the 5′ terminus or an internal site, mRNA decay occurs at diverse rates that are transcript specific and governed by RNA sequence and structure, translating ribosomes, and bound sRNAs or proteins. In response to environmental cues, bacteria are able to orchestrate widespread changes in mRNA lifetimes by modulating the concentration or specific activity of cellular ribonucleases or by unmasking the mRNA-degrading activity of cellular toxins.

The functional efficacy of the HIV-1 Rev protein is highly dependent on its ability to assemble onto its HIV-1 RNA target (the RRE) as a multimeric complex. To elucidate the mechanism of multimeric assembly, we have devised two rapid and broadly applicable strategies for examining cooperative interactions between proteins bound to RNA, one based on cooperative translational repression of a two-site reporter and the other on gel shift analysis with crude E. coli extracts. Using these strategies, we have identified two distinct surfaces of Rev (head and tail) that are critical for different steps in multimeric assembly. Our data indicate that Rev assembles cooperatively on the RRE via a series of symmetrical tail-to-tail and head-to-head protein–protein interactions. The insights into molecular architecture suggested by these findings have enabled us to derive a structural model for Rev and its multimerization on the RRE.

RNase E is a key regulatory enzyme that controls the principal pathway for mRNA degradation in Escherichia coli. The cellular concentration of this endonuclease is governed by a feedback mechanism in which RNase E tightly regulates its own synthesis. Autoregulation is mediated in cis by the 361-nucleotide 5′ untranslated region (UTR) of rne (RNase E) mRNA. Here we report the determination of the secondary structure of the rne 5′ UTR by phylogenetic comparison and chemical alkylation, together with dissection studies to identify the 5′ UTR element that mediates autoregulation. Our findings reveal that the structure and function of the rne 5′ UTRs are evolutionarily well conserved despite extensive sequence divergence. Within the rne 5′ UTRs are multiple RNA secondary structure elements, two of which function incis to mediate feedback regulation of rne gene expression. The more potent of these two elements is a stem–loop structure containing an internal loop whose sequence is the most highly conserved of any region of the rne 5′ UTR. Our data show that this stem–loop functions as a sensor of cellular RNase E activity that directs autoregulation by modulating the degradation rate ofrne mRNA in response to changes in RNase E activity.

RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli . Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5′ end. Here we show that the preference of E. coli RNase E for 5′-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related E. coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis , indicating that the 5′-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5′-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E. coli . Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E. coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.

In this issue of Molecular Cell, Bouvier et al. (2008) show that bacterial sRNAs can repress mRNA translation not only by binding to the Shine-Dalgarno element but also by base pairing anywhere within the first few codons of the protein-coding region.

Although the field of RNA biology can trace its origin to the early 20th century, it first achieved prominence in the decade from 1955 to 1965 with pivotal breakthroughs that included the discovery of ribosomes, tRNA, and mRNA and the cracking of the genetic code. The next 30 years witnessed many other seminal discoveries, such as mRNA splicing, catalytic RNA, and regulatory RNA. To meet the needs of this flourishing field of investigation, those four decades concluded 20 years ago with the founding of the RNA Society and its monthly journal RNA. Progress in the field since then is well illustrated by advances in any number of subdisciplines. Consider the two principal fates of mRNA: translation and degradation. By 1990, much was understood about translation in bacterial and eukaryotic cells, despite the dearth of structural detail at that time. In contrast, descriptions of mRNA degradation were still largely phenomenological, even in so well studied an organism as Escherichia coli. By then, it had been established that the lifetimes of bacterial mRNAs, and even of distinct segments within the same transcript, can differ significantly from one another and that such differences in longevity can have a profound effect on gene expression. However, the basis for these differences remained a mystery, as emerging evidence that bacterial mRNA lifetimes might be governed by as yet uncharacterized features of the 5′ untranslated region was difficult to reconcile with the apparent absence of 5′ exoribonuclease activity in E. coli, whose assortment of ribonucleases is limited to endonucleases and 3′ exonucleases. Because those 3′ exonucleases were known to be impeded upon encountering base-paired regions, such as the stem–loop structures typically present at the 3′ end of bacterial mRNAs, it was hypothesized that RNA degradation in bacterial cells generally begins with internal cleavage by a low-specificity endonuclease to generate a 5′ fragment susceptible to attack by 3′ exonucleases, such as polynucleotide phosphorylase (PNPase) and RNase II, and a 3′ fragment presumed to undergo further endonucleolytic fragmentation followed by exonucleolytic digestion. However, this rudimentary mechanism provided no clue as to the identity of the endonuclease, nor did it explain how base-paired regions ever get degraded or why the 3′ product of the initial endonucleolytic event is typically shorter lived (i.e., more susceptible to cleavage) than its intact precursor. The early 1990s witnessed some key discoveries, including the identification of RNase E, which targets single-stranded regions, as the endonuclease most important for mRNA degradation in E. coli and the observation that it forms a multisubunit complex (the RNA degradosome) with PNPase and other proteins. Concurrent studies showed that base-pairing at the 5′ terminus can prolong the lifetime of RNA in E. coli by impeding internal cleavage by RNase E, a counterintuitive finding that corroborated the influence of the 5′ untranslated region on transcript stability without suggesting a mechanism. In addition, poly(A) polymerase, an enzyme activity long known to exist in bacteria but largely ignored, perhaps because of the ephemeral nature of bacterial poly(A) tails, was found to destabilize certain RNA decay intermediates by an unknown mechanism. This was roughly the state of knowledge about bacterial mRNA degradation when the first issue of RNA was published 20 years ago—a lot of phenomenology but little real understanding. What followed was a profusion of mechanistic insights, beginning with revelations about degradative events at the 3′ end. Among the first was the discovery that poly(A)polymerase facilitates the degradation of decay intermediates that end with a stem–loop by adding a 3′-terminal single-stranded tail that PNPase and the RNA helicase RhlB (another degradosome component) can use as a beachhead for launching an assault on the stem–loop preceding it. At last, a mechanism for degrading base-paired regions of RNA was in hand. Interestingly, due to the reversibility of RNA phosphorolysis, PNPase was found not only to degrade RNA in bacteria but also to add heteropolymeric tails that are A-rich, enabling this enzyme to single-handedly create unstructured 3′ ends on which it can reverse course and launch an exonucleolytic attack. In contrast, the hydrolytic 3′ exonuclease RNase II, which does not associate with an E. coli helicase, proved unexpectedly to have a protective rather than a destructive effect on base-paired 3′ ends due to its propensity to selectively remove poly(A) tails without damaging the 3′ stem–loop. Furthermore, the discovery of tmRNA-mediated trans-translation explained what happens when 3′ exonucleases advancing toward the 5′ end encounter translating ribosomes moving in the opposite direction. By releasing ribosomes trapped at the 3′ end of mRNA decay intermediates that lack a stop codon, tmRNA was found to unmask those ends and render them susceptible to 3′-exonucleolytic attack. Important progress toward understanding the role of 5′ ends in bacterial mRNA degradation soon followed. A key insight came from the surprising discovery in 1998 that the rate at which RNase E cleaves RNA at internal sites is greatly influenced by the number of phosphates at the 5′ end, monophosphorylated substrates being cut as much as 10–100 times faster than their triphosphorylated equivalents. This unprecedented property, which was later shown to be a consequence of a discrete 5′-monophosphate-binding pocket on the surface of this endonuclease, helped to explain the very short lifetime of most 3′-terminal decay intermediates, which generally bear a single phosphate at the 5′ end. However, this property alone was insufficient to account for the influence of 5′-terminal base-pairing on the longevity of primary transcripts, whose triphosphorylated 5′ ends cannot interact with RNase E. The explanation for the latter phenomenon required the subsequent discovery of the RNA pyrophosphohydrolase RppH, which converts the triphosphorylated 5′ ends of primary transcripts to monophosphorylated termini that can interact productively with RNase E if single-stranded, thereby triggering the degradation of many RNAs by a 5′-end-dependent mechanism. Meanwhile, the advent of whole genome sequencing in the late 1990s revealed major disparities in the ribonucleolytic arsenals present in different bacterial species, making it clear that E. coli could not be considered a universal paradigm for bacterial mRNA degradation. For example, despite its critical role in E. coli, where it is essential for cell growth, RNase E proved to be absent from many other bacterial species, including Bacillus subtilis and a number of important human pathogens. Instead, those organisms were found to contain alternative ribonucleases not present in E. coli, such as RNase Y (a low-specificity endonuclease) and RNase J. The subsequent discovery that RNase J is a 5′-monophosphate-dependent 5′ exonuclease explained the rapid degradation of monophosphorylated decay intermediates in species that lack RNase E as well as the polar influence of stalled ribosomes, which had previously been shown to selectively stabilize the RNA segment downstream from the stall site in B. subtilis. In recent years, considerable progress has also been made toward understanding the influence of ancillary factors on bacterial mRNA degradation, most notably the effect of noncoding RNAs that base-pair with mRNA. Though first discovered in the 1980s, the widespread influence of partially complementary small RNAs (sRNAs) and fully complementary antisense RNAs was not appreciated until many more of them were identified, beginning in the mid-1990s. It soon became clear that noncoding RNAs can either down-regulate or up-regulate gene expression, frequently by controlling the accessibility of ribosome binding sites in the mRNAs to which they bind but also by altering rates of mRNA decay. Often the influence on mRNA decay is a secondary consequence of the effect on translation, due to diminished or increased protection of the mRNA by ribosomes. However, sometimes it appears to be a direct effect of the ability of a bound regulatory RNA to create or mask an internal cleavage site or to recruit RNase E, either because the sRNA is monophosphorylated or because it is associated with Hfq, an Sm-like protein chaperone that can bind to RNase E. In the meantime, parallel studies of mRNA decay in eukaryotic cells have identified a variety of degradative pathways triggered by poly(A) tail removal, decapping, or endonucleolytic cleavage. Some of these eukaryotic pathways and the enzymes on which they rely have counterparts in bacteria, suggesting the divergent evolution of ancient RNA degradation mechanisms that date back to a common progenitor. As much as has been learned about mRNA degradation in the last 20 years, even more is not yet understood, and the next 20 years of progress in elucidating the mechanisms and biological impact of this critical regulatory process in each of the three domains of life promise to be fascinating. More generally, what do the coming decades have in store for the field of RNA biology as a whole? The essence of hypothesis-driven scientific investigation is to resolve known unknowns, which by their nature are definable if countless. As for unknown unknowns, there surely are many of those as well, RNA biology having repeatedly been reinvigorated by unanticipated discoveries that have enabled the field to reinvent itself. Find a seat. The sequel is about to begin.

The spliceosomal proteins U1A and U2B" each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpll and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B" to U2hpIV is facilitated by the ancillary protein U2A', whereas specific binding of U1A to U1hpll requires no cofactor. Here we report that U2A' enables U2B" to distinguish the loop sequence of U2hpIV from that of U1hpll but plays no role in stem sequence discrimination. Although U2A' can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the approximately 500-fold greater affinity of U2A' for U2B". Additional experiments have identified a single leucine residue in U1A(Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1 hpll in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B" can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A' to the specificity of U2B".

Novel RNA-binding proteins with customized specificities can be isolated by genetic selection from combinatorial libraries. Such proteins have great potential as agents for targeted manipulation of gene expression.

A broadly applicable genetic strategy was developed for investigating RNA–protein interactions and applied to the HIV-1 Rev protein. By rapidly screening thousands of Rev–RNA interactions in Escherichia coli, we isolated Rev suppressor mutations that alleviated the deleterious effect of mutations in RRE stem–loop IIB, the high affinity RNA-binding site for Rev. All of these suppressor mutations map to a single arginine-deficient face of a Rev α-helix, and some alter the binding specificity of the protein, providing genetic evidence for direct contacts between specific Rev amino acids and RNA nucleotides in the RNA complex of Rev. The spatial constraints suggested by these data have enabled us to model the structure of this complex.