Mini-III, a fourth class of RNase III catalyses maturation of the Bacillus subtilis 23S ribosomal RNA

Ribonuclease III (RNase III) type of enzymes are double-stranded RNA (dsRNA)-specific endoribonucleases that have important roles in RNA maturation and mRNA decay. They are involved in processing precursors of ribosomal RNA (rRNA) in bacteria as well as precursors of short interfering RNAs (siRNAs) and microRNAs (miRNAs) in eukaryotes. RNase III proteins have been grouped in three major classes according to their domain organization. In this issue of Molecular Microbiology, Redko et al. identified a novel class of bacterial RNase III, named Mini-III, consisting only of the RNase III catalytic domain and functioning in the maturation of the 23S rRNA in Bacillus subtilis. Its absence from proteobacteria reveals that this step is mechanistically different from the corresponding step in Escherichia coli. The fact that Mini-III orthologues are present in unicellular photosynthetic eukaryotes and in plants opens new opportunities for functional studies of this type of RNases.     

Alpha-hydroxytropolones are noncompetitive inhibitors of human RNase H1 that bind to the active site and modulate substrate binding

The ribonucleases H (RNases H) of HIV and hepatitis B virus are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. Eukaryotic ribonuclease H1 (RNase H1) is an essential protein and a probable off-target enzyme for viral RNase H inhibitors. α-hydroxytropolones (αHTs) are a class of anti-RNase H inhibitors that can inhibit the HIV, hepatitis B virus, and human RNases H1; however, it is unclear how these inhibitors could be developed to distinguish between these enzymes. To accelerate the development of selective RNase H inhibitors, we performed biochemical and kinetic studies on the human enzyme, which was recombinantly expressed in Escherichia coli. Size-exclusion chromatography showed that free RNase H1 is monomeric and forms a 2:1 complex with a substrate of 12 bp. FRET heteroduplex cleavage assays were used to test inhibition of RNase H1 in steady-state kinetics by two structurally diverse αHTs, 110 and 404. We determined that turnover rate was reduced, but inhibition was not competitive with substrate, despite inhibitor binding to the active site. Given the compounds’ reversible binding to the active site, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which, by binding to the active site, αHTs stabilize an inactive enzyme–substrate–inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid the development of RNase H inhibitors selective for the viral enzymes.     

Degradation of double-stranded RNA by human pancreatic ribonuclease: crucial role of noncatalytic basic amino acid residues

Under physiological salt conditions double-stranded (ds) RNA is resistant to the action of most mammalian extracellular ribonucleases (RNases). However, some pancreatic-type RNases are able to degrade dsRNA under conditions in which the activity of bovine RNase A, the prototype of the RNase superfamily, is essentially undetectable. Human pancreatic ribonuclease (HP-RNase) is the most powerful enzyme to degrade dsRNA within the tetrapod RNase superfamily, being 500-fold more active than the orthologous bovine enzyme on this substrate. HP-RNase has basic amino acids at positions where RNase A shows instead neutral residues. We found by modeling that some of these basic charges are located on the periphery of the substrate binding site. To verify the role of these residues in the cleavage of dsRNA, we prepared four variants of HP-RNase: R4A, G38D, K102A, and the triple mutant R4A/G38D/K102A. The overall structure and active site conformation of the variants were not significantly affected by the amino acid substitutions, as deduced from CD spectra and activity on single-stranded RNA substrates. The kinetic parameters of the mutants with double-helical poly(A).poly(U) as a substrate were determined, as well as their helix-destabilizing action on a synthetic DNA substrate. The results obtained indicate that the potent activity of HP-RNase on dsRNA is related to the presence of noncatalytic basic residues which cooperatively contribute to the binding and destabilization of the double-helical RNA molecule. These data and the wide distribution of the enzyme in different organs and body fluids suggest that HP-RNase has evolved to perform both digestive and nondigestive physiological functions.     

Defining the enzyme binding domain of a ribonuclease III processing signal. Ethylation interference and hydroxyl radical footprinting using catalytically inactive RNase III mutants.

Ethylation interference and hydroxyl radical footprinting were used to identify substrate ribose-phosphate backbone sites that interact with the Escherichia coli RNA processing enzyme, ribonuclease III. Two RNase III mutants were employed, which bind substrate in vitro similarly as wild-type enzyme, but lack detectable phosphodiesterase activity. Specifically, altering glutamic acid at position 117 to lysine or alanine uncouples substrate binding from cleavage. The two substrates examined are based on the bacteriophage T7 R1.1 RNase III processing signal. One substrate, R1.1 RNA, undergoes accurate single cleavage at the canonical site, while a close variant, R1.1[WC-L] RNA, undergoes coordinate double cleavage. The interference and footprinting patterns for each substrate (i) overlap, (ii) exhibit symmetry and (iii) extend approximately one helical turn in each direction from the RNase III cleavage sites. Divalent metal ions (Mg2+, Ca2+) significantly enhance substrate binding, and confer stronger protection from hydroxyl radicals, but do not significantly affect the interference pattern. The footprinting and interference patterns indicate that (i) RNase III contacts the sugar-phosphate backbone; (ii) the RNase III-substrate interaction spans two turns of the A-form helix; and (iii) divalent metal ion does not play an essential role in binding specificity. These results rationalize the conserved two-turn helix motif seen in most RNase III processing signals, and which is necessary for optimal processing reactivity. In addition, the specific differences in the footprint and interference patterns of the two substrates suggest why RNase III catalyzes the coordinate double cleavage of R1.1[WC-L] RNA, and dsRNA in general, while catalyzing only single cleavage of R1.1 RNA and related substrates in which the scissle bond is within an asymmetric internal loop.     

Short RNA duplexes guide sequence-dependent cleavage by human Dicer

Dicer is a member of the double-stranded (ds) RNA-specific ribonuclease III (RNase III) family that is required for RNA processing and degradation. Like most members of the RNase III family, Dicer possesses a dsRNA binding domain and cleaves long RNA duplexes in vitro. In this study, Dicer substrate selectivity was examined using bipartite substrates. These experiments revealed that an RNA helix possessing a 2-nucleotide (nt) 3'-overhang may bind and direct sequence-specific Dicer-mediated cleavage in trans at a fixed distance from the 3'-end overhang. Chemical modifications of the substrate indicate that the presence of the ribose 2'-hydroxyl group is not required for Dicer binding, but some located near the scissile bonds are needed for RNA cleavage. This suggests a flexible mechanism for substrate selectivity that recognizes the overall shape of an RNA helix. Examination of the structure of natural pre-microRNAs (pre-miRNAs) suggests that they may form bipartite substrates with complementary mRNA sequences, and thus induce seed-independent Dicer cleavage. Indeed, in vitro, natural pre-miRNA directed sequence-specific Dicer-mediated cleavage in trans by supporting the formation of a substrate mimic.     

Site-Specific RNase A Activity Was Dramatically Reduced in Serum from Multiple Types of Cancer Patients

Potent RNase activities were found in the serum of mammals but the physiological function of the RNases was never well illustrated, largely due to the caveats in methods of RNase activity measurement. None of the existing methods can distinguish between RNases with different target specificities. A systematic study was recently carried out in our lab to investigate the site-specificity of serum RNases on double-stranded RNA substrates, and found that serum RNases cleave double-stranded RNAs predominantly at 5′-U/A-3′ and 5′-C/A-3′ dinucleotide sites, in a manner closely resembling RNase A. Based on this finding, a FRET assay was developed in the current study to measure this site-specific serum RNase activity in human samples using a double stranded RNA substrate. We demonstrated that the method has a dynamic range of 10⁻⁵ mg/ml- 10⁻¹ mg/ml using serial dilution of RNase A. The sera of 303 cancer patients were subjected to comparison with 128 healthy controls, and it was found that serum RNase activities visualized with this site-specific double stranded probe were found to be significantly reduced in patients with gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovary cancer, cervical cancer, bladder cancer, kidney cancer and lung cancer, while only minor changes were found in breast and colon cancer patients. This is the first report using double stranded RNA as probe to quantify site-specific activities of RNase A in a serum. The results illustrated that RNase A might be further evaluated to determine if it can serve as a new class of biomarkers for certain cancer types.     

Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of a mRNA containing the same sequence. The 21–23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that dsRNA specifically suppresses the expression of HIV-1 genes. To study dsRNA-mediated gene interference in HIV-1-infected cells, we have designed six long dsRNAs containing the HIV-1 gag and env genes. HIV-1 replication was totally suppressed in a sequence-specific manner by the dsRNAs in HIV-1-infected cells. Especially, E2 dsRNA containing the major CD4-binding domain sequence of gp120, as the target of the HIV-1 env gene, dramatically inhibited the expression of the HIV-1 p24 antigen in PBMCs for a relatively long time. The dsRNA interference method seems to be a promising new strategy for anti-HIV-1 gene therapeutics.     

Ribonucleases J1 and J2: two novel endoribonucleases in B.subtilis with functional homology to E.coli RNase E

Many prokaryotic organisms lack an equivalent of RNase E, which plays a key role in mRNA degradation in Escherichia coli. In this paper, we report the purification and identification by mass spectrometry in Bacillus subtilis of two paralogous endoribonucleases, here named RNases J1 and J2, which share functional homologies with RNase E but no sequence similarity. Both enzymes are able to cleave the B.subtilis thrS leader at a site that can also be cleaved by E.coli RNase E. We have previously shown that cleavage at this site increases the stability of the downstream messenger. Moreover, RNases J1/J2 are sensitive to the 5′ phosphorylation state of the substrate in a site-specific manner. Orthologues of RNases J1/J2, which belong to the metallo-β-lactamase family, are evolutionarily conserved in many prokaryotic organisms, representing a new family of endoribonucleases. RNases J1/J2 appear to be implicated in regulatory processing/maturation of specific mRNAs, such as the T-box family members thrS and thrZ, but may also contribute to global mRNA degradation.     

Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p

Members of the double-stranded RNA (dsRNA) specific RNase III family are known to use a conserved dsRNA-binding domain (dsRBD) to distinguish RNA A-form helices from DNA B-form ones, however, the basis of this selectivity and its effect on cleavage specificity remain unknown. Here, we directly examine the molecular requirements for dsRNA recognition and cleavage by the budding yeast RNase III (Rnt1p), and compare it to both bacterial RNase III and fission yeast RNase III (Pac1). We synthesized substrates with either chemically modified nucleotides near the cleavage sites, or with different DNA/RNA combinations, and investigated their binding and cleavage by Rnt1p. Substitution for the ribonucleotide vicinal to the scissile phosphodiester linkage with 2'-deoxy-2'-fluoro-beta-d-ribose (2' F-RNA), a deoxyribonucleotide, or a 2'-O-methylribonucleotide permitted cleavage by Rnt1p, while the introduction of a 2', 5'-phosphodiester linkage permitted binding, but not cleavage. This indicates that the position of the phosphodiester link with respect to the nuclease domain, and not the 2'-OH group, is critical for cleavage by Rnt1p. Surprisingly, Rnt1p bound to a DNA helix capped with an NGNN tetraribonucleotide loop indicating that the binding of at least one member of the RNase III family is not restricted to RNA. The results also suggest that the dsRBD may accommodate B-form DNA duplexes. Interestingly, Rnt1p, but not Pac1 nor bacterial RNase III, cleaved the DNA strand of a DNA/RNA hybrid, indicating that A-form RNA helix is not essential for cleavage by Rnt1p. In contrast, RNA/DNA hybrids bound to, but were not cleaved by Rnt1p, underscoring the critical role for the nucleotide located at 3' end of the tetraloop and suggesting an asymmetrical mode of substrate recognition. In cell extracts, the native enzyme effectively cleaved the DNA/RNA hybrid, indicating much broader Rnt1p substrate specificity than previously thought. The discovery of this novel RNA-dependent deoxyribonuclease activity has potential implications in devising new antiviral strategies that target actively transcribed DNA.     

Ethidium-dependent uncoupling of substrate binding and cleavage by Escherichia coli ribonuclease III

Ethidium bromide (EB) is known to inhibit cleavage of bacterial rRNA precursors by Escherichia coli ribonuclease III, a dsRNA-specific nuclease. The mechanism of EB inhibition of RNase III is not known nor is there information on EB-binding sites in RNase III substrates. We show here that EB is a reversible, apparently competitive inhibitor of RNase III cleavage of small model substrates in vitro. Inhibition is due to intercalation, since (i) the inhibitory concentrations of EB are similar to measured EB intercalation affinities; (ii) substrate cleavage is not affected by actinomycin D, an intercalating agent that does not bind dsRNA; (iii) the EB concentration dependence of inhibition is a function of substrate structure. In contrast, EB does not strongly inhibit the ability of RNase III to bind substrate. EB also does not block substrate binding by the C-terminal dsRNA-binding domain (dsRBD) of RNase III, indicating that EB perturbs substrate recognition by the N-terminal catalytic domain. Laser photocleavage experiments revealed two ethidium-binding sites in the substrate R1.1 RNA. One site is in the internal loop, adjacent to the scissile bond, while the second site is in the lower stem. Both sites consist of an A-A pair stacked on a CG pair, a motif which apparently provides a particularly favorable environment for intercalation. These results indicate an inhibitory mechanism in which EB site-specifically binds substrate, creating a cleavage-resistant complex that can compete with free substrate for RNase III. This study also shows that RNase III recognition and cleavage of substrate can be uncoupled and supports an enzymatic mechanism of dsRNA cleavage involving cooperative but not obligatorily linked actions of the dsRBD and the catalytic domain.     

Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro

Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a k(cat) value that is one-half that of wt RNase III, but exhibits an unaltered K(m). Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg(2+) ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0+/-0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg(2+)-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively.     

RNase L releases a small RNA from HCV RNA that refolds into a potent PAMP

Triggering and propagating an intracellular innate immune response is essential for control of viral infections. RNase L is a host endoribonuclease and a pivotal component of innate immunity that cleaves viral and cellular RNA within single-stranded loops releasing small structured RNAs with 5′-hydroxyl (5′-OH) and 3′-monophosphoryl (3′-p) groups. In 2007, we reported that RNase L cleaves self RNA to produce small RNAs that function as pathogen-associated molecular patterns (PAMPs). However, the precise sequence and structure of PAMP RNAs produced by RNase L is unknown. Here we used hepatitis C virus RNA as substrate to characterize RNase L mediated cleavage products [named suppressor of virus RNA (svRNA)] for their ability to activate RIG-I like receptors (RLR). The NS5B region of HCV RNA was cleaved by RNase L to release an svRNA that bound to RIG-I, displacing its repressor domain and stimulating its ATPase activity while signaling to the IFN-β gene in intact cells. All three of these RIG-I functions were dependent on the presence in svRNA of the 3′-p. Furthermore, svRNA suppressed HCV replication in vitro through a mechanism involving IFN production and triggered a RIG-I-dependent hepatic innate immune response in mice. RNase L and OAS (required for its activation) were both expressed in hepatocytes from HCV-infected patients, raising the possibility that the OAS/RNase L pathway might suppress HCV replication in vivo. It is proposed that RNase L mediated cleavage of HCV RNA generates svRNA that activates RIG-I, thus propagating innate immune signaling to the IFN-β gene.     

Sequence dependence of substrate recognition and cleavage by yeast RNase III

Yeast Rnt1p is a member of the double-stranded RNA (dsRNA) specific RNase III family of endoribonucleases involved in RNA processing and RNA interference (RNAi). Unlike other RNase III enzymes, which recognize a variety of RNA duplexes, Rnt1p cleaves specifically RNA stems capped with the conserved AGNN tetraloop. This unusual substrate specificity challenges the established dogma for substrate selection by RNase III and questions the dsRNA contribution to recognition by Rnt1p. Here we show that the dsRNA sequence adjacent to the tetraloop regulates Rnt1p cleavage by interfering with RNA binding. In context, sequences surrounding the cleavage site directly influence the cleavage efficiency. Introduction of sequences that stabilize the RNA helix enhanced binding while reducing the turnover rate indicating that, unlike the tetraloop, Rnt1p binding to the dsRNA helix may become rate-limiting. These results suggest that Rnt1p activity is strictly regulated by a combination of primary and tertiary structural elements allowing a substrate-specific binding and cleavage efficiency.     

Evidence for recycling of external guide sequences during cleavage of bipartite substrates in vitro by reconstituted archaeal RNase P

RNA-mediated RNA cleavage events are being increasingly exploited to disrupt RNA function, an important objective in post-genomic biology. RNase P, a ribonucleoprotein enzyme that catalyzes the removal of 5'-leaders from precursor tRNAs, has previously been utilized for sequence-specific cleavage of cellular RNAs. In one of these strategies, borne out in bacterial and mammalian cell culture, an external guide sequence (EGS) RNA base-paired to a target RNA makes the latter a substrate for endogenous RNase P by rendering the bipartite target RNA-EGS complex a precursor tRNA structural mimic. In this study, we first obtained evidence that four different mesophilic and thermophilic archaeal RNase P holoenzymes, reconstituted in vitro using their respective constituent RNA and protein subunits, recognize and cleave such substrate-EGS complexes. We further demonstrate that these EGSs engage in multiple rounds of substrate recognition while assisting archaeal RNase P-mediated cleavage of a target RNA in vitro. Taken together, the EGS-based approach merits consideration as a gene knockdown tool in archaea.     

A ribonuclease III involved in virulence of Mucorales fungi has evolved to cut exclusively single-stranded RNA

Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition.     

Sequence-specific cleavage of the RNA strand in DNA–RNA hybrids by the fusion of ribonuclease H with a zinc finger

Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. Here, we report the development of a new enzyme that cleaves the RNA strand in DNA–RNA hybrids 5 nt from a nonanucleotide recognition sequence. The enzyme was constructed by fusing two functionally independent domains, a RNase HI, that hydrolyzes RNA in DNA–RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA–RNA hybrids. The optimization of the fusion enzyme’s specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.     

Double-stranded RNA Mediates Selective Gene Silencing of Protein Phosphatase Type 1 Delta Isoform in HEK-293 Cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1δ) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP18. This RNA interference did not affect the expression of α and γ1 isoforms of PP1. Transfection of antisense RNA specific for PP1δ also suppressed the expression of PP1δ. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.     

The non-RNase H domain of Saccharomyces cerevisiae RNase H1 binds double-stranded RNA: magnesium modulates the switch between double-stranded RNA binding and RNase H activity.

Eukaryotic ribonucleases H of known sequence are composed of an RNase H domain similar in size and sequence to that of Escherichia coli RNase HI and additional domains of unknown function. The RNase H1 of Saccharomyces cerevisiae has such an RNase H domain at its C-terminus. Here we show that the N-terminal non-RNase H portion of the yeast RNase H1 binds tightly to double-stranded RNA (dsRNA) and RNA-DNA hybrids even in the absence of the RNase H domain. Two copies of a sequence with limited similarity to the dsRNA-binding motif are present in this N-terminus. When the first of these sequences is altered, the protein no longer binds tightly to dsRNA and exhibits an increase in RNase H activity. Unlike other dsRNA-binding proteins, increasing the Mg2+ concentration from 0.5 mM to 5 mM inhibits binding of RNase H1 to dsRNA; yet a protein missing the RNase H domain binds strongly to dsRNA even at the higher Mg2+ concentration. These results suggest that binding to dsRNA and RNase H activity are mutually exclusive, and the Mg2+ concentration is critical for switching between the activities. Changes in the Mg2+ concentration or proteolytic severing of the dsRNA-binding domain could alter the activity or location of the RNase H and may govern access of the enzyme to the substrate. Sequences similar to the dsRNA-binding motif are present in other eukaryotic RNases H and the transactivating protein of cauliflower mosaic virus, suggesting that these proteins may also bind to dsRNA.