RNAse P

The delivery of a macromolecule to the cytosol of human cells is assessed by using a pendant di-O-glycosylated derivative of fluorescein. Its fluorescence is unmasked by Escherichia coli βgalactosidase installed in the cytosol. Background is diminished by using RNAi to suppress the expression of GLB1, which encodes a lysosomal β-galactosidase. This strategy was used to quantify the cytosolic entry of a highly cationic protein, ribonuclease A. The cytosol of mammalian cells is the intended destination for many proteins and other macromolecules of interest. 1 Typically, delivery entails endocytosis, followed by translocation from an endosomal compartment to the cytosol. Uptake into endosomes can be facilitated by a cationic appendage 2 or graft, and can be assessed with a pendant fluorogenic probe that is unmasked by an endosomal esterase. But it is the next step, entailing traversal of a phospholipid bilayer, that is the major hurdle to cytosolic delivery. 9 Endosome-to-cytosol translocation is a rare event and thus difficult to assess. Two strategies are evident for distinguishing the cytosolic pool of internalized molecules from the endosomal pool. One is to separate membrane-bound compartments from the cytosol by subcellular fractionation. This method requires cell lysis, and is thus inapplicable to live cells or high-throughput screens. A second method relies on the modification of a latent probe, such that macromolecules that reach the cytosol are differentiated from those in endosomes. Previous strategies for measuring translocation to the cytosol have relied on fluorescence resonance energy transfer (FRET), either by monitoring FRET between a preinstalled cytosolic fluorescent protein and a fluorophore-tagged peptide, 12 or by monitoring the unquenching of fluorescence of a pair of disulfide-linked fluorophores. Although these strategies did overcome the difficulty of excluding signals from endocytic compartments, they suffer from a high background. To avoid the background that accompanies chemical reactions, we sought an enzymesubstrate pair that was orthogonal to human cells. We reasoned that macromolecules tagged with fluorescein di-β-D-galactopyranoside (FDG) could be unmasked by Escherichia coli βgalactosidase (β-gal) produced by heterologous expression in the cytosol (Fig. ). As a † Electronic supplementary information (ESI) available: Experimental procesures. See

The nucleoprotein (N) of measles virus encapsidates viral genomic RNA to form a helical nucleocapsid. Its strong self-association is a major hurdle in determining its high-resolution structure using X-ray crystallography. We report the bacterial expression, purification, and characterization of a variant N that has lost its ability to form nucleocapsid-like structures after substitution of two residues by polar residues. Using immunoprecipitation, circular dichroism, and limited proteolysis studies, we show that this nucleoprotein retains a folding similar to wild-type N. Furthermore, the variant N binds the phosphoprotein, indicating that it retains biochemical relevance. We also present evidence indicating that the N-terminus of N lies at the surface of the nucleocapsid. Beyond the identification of one region of N involved in self-association, our results should facilitate structural studies of N using X-ray crystallography.

There is considerable interest in the discovery of peptide ligands that bind to protein targets. Discovery of such ligands is usually approached by screening large peptide libraries. However, the individual peptides must be tethered to a tag that preserves their individual identities (e.g., phage display or one-bead one-compound). To overcome this limitation, we have developed a method for screening libraries of label-free peptides for binding to a protein target in solution as a single batch. The screening is based on decreased amide hydrogen exchange by peptides that bind to the target. Hydrogen exchange is measured by mass spectrometry. We demonstrate the approach using a peptide library derived from the Escherichia coli proteome that contained 6664 identifiable features. The library was spiked separately with a peptide spanning the calmodulin binding domain of endothelial nitric oxide synthase (eNOS, 494-513) and a peptide spanning the N-terminal 20 residues of bovine ribonuclea...

Ribosome biogenesis and turnover are processes necessary for cell viability and proliferation, and many kinds of proteins are known to regulate these processes. However, many questions still remain, especially in the Archaea. Generally, several ribonucleases are required to process precursor rRNAs to their mature forms, and to degrade rRNAs for quality control. Here, we found that FAU-1, which is known to be an RNA binding protein, possesses an RNase activity against precursor 5S rRNA derived from P. furiosus and T. kodakarensis in the order Thermococcales in vitro. An in vitro analysis revealed that UA sequences in the upstream of 5S rRNA were preferentially degraded by addition of FAU-1. Moreover, a fau-1 gene deletion mutant of T. kodakarensis showed a delay of exponential phase, reduction of maximum cell number and significant changes in the nucleotide sequence lengths of its 5S, 16S, and 23S rRNAs in early exponential phase. Our results suggest that FAU-1 is a potential RNase involved in rRNA stability through maturation and/or degradation processes.

Highlights d Endolysosomal RNase 2 and RNase T2 cooperatively process RNA into TLR8 ligands d RNase 2 and RNase T2 synergistically release uridine from RNA ligands d RNase T2-hypomorphic patients have an impaired to TLR8 response to pathogen RNA d Sensing of live pathogens by TLR8 requires both RNase 2 and RNase T2

Bovine seminal ribonuclease, the only di- meric ribonuclease described thus far, is found to exist in two different quaternary structure forms. In one, the N-terminal segment (residues 1-17) of each subunit is interchanged with the remaining segment of the other subunit, whereas in the second, such interchange does not occur. Functionally, they differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity. Each form can convert into the other, up to an equilibrium ratio, which is that found for the isolated protein. The results of refolding experiments of unfolded protein chains suggest that also in vivo the form lacking interchange may be produced first and is then partially trans- formed into the other dimeric form until equilibrium is reached. Although the implications of these findings may not be immediately apparent, they are intriguing and may have an impact on the unusual noncatalytic actions of the protein, such as its selective cytotoxicity toward tumor cells, activated T cells, and differentiated male germ cells. Bovine seminal ribonuclease (BS-RNase) is a homodimeric ribonuclease-in fact, the only dimeric RNase isolated thus far. Its enzymic properties are unusual for a ribonuclease, as it cleaves effectively singleand double-stranded RNA and is allosterically regulated in the rate-determining step of the reaction. These properties are matched by its unusual biological, noncatalytic actions, including its selective toxicity toward tumor cells, activated T cells, and the male germ cell line (ref. 1 and references cited therein). In the structure of naturally dimeric BS-RNase determined by x-ray crystallography (2, 3), the two subunits interchange their N-terminal segments (see Fig. ) as do the monomers of artificially dimerized bovin' pancreatic RNase (RNase A) in the structure proposed by Crestfield and others (4, 5). A significant consequeoce of ths 'structural arrangement is the composite nature 9f the active sites of dimerized RNase A and of naturally dimeric BS-RNase. Composite, shared active sites have been previously found for a few oligomeric enzymes (6-9). However, in all of these cases, dissociation of the oligomers produced inactive mono-

The characteristics and mode of action of a single-strand-specific nuclease isolated from rat liver endoplasmic reticulum are investigated with respect to its DNA and RNA substrates. 2. The RNase activity of the enzyme is slightly influenced by the presence of divalent cations but the DNase activity is enhanced by divalent cations particularly Mn 2+. 3. Activity is partially inhibited by the presence of EGTA; this effect is reversed most efficiently by the addition of Mn 2+. 4. The enzyme exhibits small pH dependence between pH 6-9 and maximum activity is observed at pH 7-7.5 for both DNase and RNase activities. 5. Sulfhydryl group reagents do not affect its action but histidyl group reagents exert a small but definite effect. 6. The enzyme degrades DNA and RNA endonucleolyticaHy producing fragments which possess Y-OH and 5'-phosphate termini. 7. Monomers arc not produced even after prolonged degradation. 8. The end product of poly(U)degradation ranges between two and four building blocks but the DNA product is longer probably due to considerable percentage of secondary structure.

An endonuclease, which was originally identified for its RNA polymerase inhibitory activity, was isolated from rat liver endoplasmic reticulum. The enzyme yields on gel chromatography four active fractions of different molecular weights ( M , 5.3 x lo4, 9 x lo4, 1.55 x lo5 and Sephacryl S-200 fraction at Vo). Each fraction contains polypeptide chains which give a single band on sodium dodecylsulphate electrophoresis ( M , 5.4 x 10"). This indicates that the enzyme is an oligomeric protein and each of its subunits exhibits the same or very similar molecular weights. Deoxyribonucleoside and ribonucleoside triphosphales can bind to the endoplasmic reticulum nuclease. Binding is enhanced in the presence of divalent cations particularly MgZf. The enzyme exhibits mainly RNase activity but can also degrade denatured DNA and D N A . RNA hybrids which contain breaks in one of the two strands. Poly(A) and mainly poly(U) are most susceptible to its nucleolytic activity whereas poly(C) is completely resistant

RNA Synthesis: All RNA oligonucleotides were obtained commercially from the HHMI Biopolymer/Keck Foundation Biotechnology Resource Laboratory RNA at the Yale University School of Medicine. RNA 1, the stem-loop RNA, has the sequence GA(dTF)ACGUUCGCG(dTN)AUC where dTF is a fluorescein coupled to a 2'-deoxythymidine and dTN is a 2'-deoxythymidine with a 5' C6amino linker. The unstructured RNA (RNA 2) has the sequence GU(dTF)UCGCCAUUC(dTN)AAG, with similar modified bases. RNA oligonucleotides were deprotected and labeled as previously described. 1 The acceptor fluorophore tetramethylrhodamine was post-synthetically coupled to the amino-modifier dTN as described. 1 Buffer Conditions: A near-physiological standard buffer was prepared containing 130 mM potassium glutamate (introducing ~100 mM K + ) and 1 mM MgCl 2 at pH 7.5. 2 DTT was added directly prior to experiments to a final concentration of 10 mM. Since the buffer capacity of glutamate is relatively low around physiological pH, we confirmed that the pH did not change by more than 0.1 pH units upon addition of all supplements to their highest concentration, including RNase T 1 (stored in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA; then diluted in standard buffer to the appropriate concentration), protease inhibitor treated S100 cytosolic extract from HeLa cells (pH 7.6; preparation see below), and acidic aurin tricarboxylic acid (ATA; added to not higher than 0.5 mM final concentration), unless otherwise noted. S100 Cytosolic Extract From HeLa Cells: This cell extract was a gift from Danny Reinberg (Department of Biochemistry, State University of New Jersey, Rutgers) and was prepared following published protocols. Final step is an extensive dialysis against buffer D, composed of 20 mM HEPES, pH 7.9, 20% (v/v) Glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF; we found the pH after dialysis to be 7.6. UV Melting Curves: 1 µM unlabeled or doubly fluorophore-labeled RNA was prepared in standard buffer and degassed under vacuum. 260 nm was used as the analytical wavelength for a UV melting experiment, and the signal at 320 nm was subtracted as background. Temperatures ramped up and down from 20 ˚C to 100 ˚C at a rate of 0.2 ˚C/minute using a Beckman DU640B Spectrophotometer with High Performance Temperature Controller and Micro Auto 6 T m cell holder. Melting temperatures were obtained using MicroCal Origin 7.0 by fitting a Gaussian distribution to the first derivative of the background-corrected 260 nm absorbance vs. temperature plot. FRET Melting Curves: Fluorescence spectra and intensities were recorded on an Aminco-Bowman Series 2 spectrofluorimeter (Thermo Spectronic). 50 nM double-labeled RNA (200 µL total volume) was prepared in standard buffer, heat-annealed at 70 ˚C for 2 minutes and centrifuge filtered through a 0.45 µm filter. Excitation (500-650 nm) and emission scans (350-570 nm) were collected at 10 nm/s and averaged over 5 repetitions, prior and after the experiment. The cuvette was sealed with Parafilm after carefully inserting a temperature microprobe such that it did not obstruct the light path. Fluorescein was excited at 490 nm (4 nm bandwidth) and fluorescence emission over time was recorded simultaneously at the fluorescein (520 nm, 8 nm bandwidth) and tetramethylrhodamine (585 nm, 8 nm bandwidth) wavelengths for a total of >11,000 sec, by shifting the emission monochromator back and forth. Over the total timeframe, the temperature was ramped from 20 ˚C to 68 ˚C (as measured with the microprobe), at steps of 1-5 ˚C. Once a new temperature became stable for over 100 s after a ramping step, FRET ratios Q = F 585 /F 520 were recorded for 100 s (at 1 datum/sec), averaged and normalized to the average FRET ratio at the lowest temperature, Q 0 (i.e., (Q-Q 0 )/Q 0 was calculated). Corrected FRET ratios were then computed by subtracting the normalized FRET ratios of stem-loop RNA 1 from the corresponding normalized FRET ratios of unstructured RNA 2 to compensate for fluorescence changes not attributed to RNA melting (but rather to direct effects of temperature on the fluorophores). The

RNase P catalyzes the 5′ maturation of transfer RNA (tRNA). RNase P from Bacillus subtilis comprises a large RNA component (130 kDa, P RNA) and a small protein subunit (14 kDa, P protein). Although P RNA alone can efficiently catalyze the maturation reaction in vitro, P protein is strictly required under physiological conditions. We have used time-resolved fluorescence resonance energy transfer on a series of donor-labeled substrates and two acceptor-labeled P proteins to determine the conformation of the pre-tRNA 5′ leader relative to the protein in the holoenzyme-pre-tRNA complex. The resulting distance distribution measurements indicate that the leader binds to the holoenzyme in an extended conformation between nucleotides 3 and 7. The conformational mobility of nucleotides 5-8 in the leader is reduced, providing further evidence that these nucleotides interact with the holoenzyme. The increased fluorescence intensity and lifetime of the 5′-fluorescein label of these leaders indicate a more hydrophobic environment, consistent with the notion that such interactions occur with the central cleft of the P protein. Taken together, our data support a model where the P protein binds to the 5′ leader between the fourth and seventh nucleotides upstream of the cleavage site, extending the leader and decreasing its structural dynamics. Thus, P protein acts as a wedge to separate the 5′ from the 3′ terminus of the pre-tRNA and to position the cleavage site in the catalytic core. These results reveal a structural basis for the P protein dependent discrimination between precursor and mature tRNAs.

Bacillus amyloliquefaciens extracellular RNase has been previously cloned and expressed in Bacillus subtilis. Site-specific mutagenesis experiments have identified codon -39 as the start site of translation. We have determined the primary signal peptide cleavage site of preprobarnase and propose a pathway for the conversion of probarnase to mature barnase.

The thermal stability of the two dimers of RNase A with N-or C-terminal swapped ends is investigated by means of dissociation kinetics, differential scanning calorimetry, and circular dichroism measurements. The data indicate that the dimer characterized by the swapping of the N-terminal a-helices is less prone to monomerize when compared to the dimer characterized by the swapping of the C-terminal h-strands. This finding is correlated to the structural features of the so-called open interface of the dimeric forms.

Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a ...

Light-activated antisense oligodeoxynucleotides (asODNs) were developed to control the degradation of target mRNA in living cells by RNase H. A 20-mer asODN previously shown to target c-myb, a hematopoietic transcription factor, was covalently attached via a photocleavable linker (PL) to partially complementary 20-mer sense strands (sODNs). In the 'caged' state, the sODN blocked hybridization of the asODN to c-myb mRNA. Six asODN-PL-sODN conjugates, C1-C6, were synthesized. C5, with twelve complementary bases, gave the largest decrease in melting temperature (T m ) upon UV irradiation ("T m = 2298C). The most thermally stable conjugate, C6 (T m = 848C), gave the lowest background RNase H activity, with just 8.6% degradation of an RNA 40-mer after 1 h incubation. In biochemical assays with C6, RNA digestion increased 10-fold 10 min after UV irradiation. Finally, phosphorothioated analogs S-C5 and S-C6 were synthesized to test activity in cultured K562 (human leukemia) cells. No knockdown of c-myb mRNA or protein was observed with intact S-C5 or S-C6, whereas more than half of c-myb mRNA was degraded 24 h after photoactivation. Two-fold photomodulation of c-MYB protein levels was also observed with S-C5. However, no photomodulation of c-MYB protein levels was observed with S-C6, perhaps due to the greater stability of this duplex.

We developed a rapid and simple method for rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems. The method relies on the sequence-specific scission of 16S rRNA with ribonuclease H (RNase H) and oligonucleotides that specifically hybridize with targeted rRNA molecules. RNAs from a complex community were first mixed with an oligonucleotide and were subsequently digested with RNase H to achieve sequence-dependent rRNA cleavage at the hybridization site. For the quantitative detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel-electrophoresis, which separated and quantified cleaved and intact rRNA fragments. This method enabled the quantitative detection of microbes in a complex microbial community by a relatively simple and fast experimental procedure. We then applied the cleavage method to actual anaerobic microbial communities such as digested sewage sludge and UASB sludges. The results demonstrated that the p...

A rapid and simple approach to the small-subunit (SSU) rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. The method employs sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H. Defined mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to as a “scissor probe”) that was specifically designed to hybridize with a particular site of targeted rRNA and were subsequently digested with RNase H to proceed to sequence-dependent rRNA scission at the hybridization site. Under appropriate reaction conditions, the targeted rRNAs were correctly cut into two fragments, whereas nontargeted rRNAs remained intact under the same conditions. The specificity of the cleavage could be properly adjusted by controlling the hybridization stringency between the rRNA and the oligonucleotides, i.e., by controlling either the temperature of the reaction or the formamide concentration in the hybridization-dig...

Vilyuisk human encephalitis virus (VHEV) is a picornavirus related to Theiler's murine encephalomyelitis virus (TMEV). VHEV was isolated from human material passaged in mice. Whether this VHEV is from human or mouse origin is therefore unclear. We took advantage of the species-specific activity of the non-structural L* protein of theiloviruses to track the origin of TMEV isolates. TMEV L* inhibits RNase L, the effector enzyme of the interferon pathway. Using co-immunoprecipitation and functional RNase L assays, the species-specificity of RNase L antagonism was tested for L* from a mouse (DA) and a rat (RTV-1) TMEV strain as well as for VHEV. Co-immunoprecipitation and functional assay data confirmed the species-specificity of L* activity and showed that L* from the rat strain RTV-1 inhibited rat but not mouse or human RNase L. Next, we showed that VHEV L* protein was phylogenetically related to L* of mouse viruses and that it failed to inhibit human RNase L but readily antagoniz...

Aim: To study the structural requirements that a cyclooligosaccharide-based nanoparticle must fulfill to be an efficient siRNA transfection vector. Materials & methods: siRNA protection from degradation by RNAses, transfection efficiency and the thermodynamic parameters of the nanoparticle/siRNA interactions were studied on pairs of amphiphilic molecules using biochemical techniques and molecular dynamics. Results: The lower the siRNA solvent accessible surface area in the presence of the nanoparticle, higher the protection from RNAse-mediated degradation in the corresponding nanocomplex; a moderate nanoparticle/siRNA binding energy value further facilitates reversible complexation and binding to the target cellular mRNA. Conclusion: The use, in advance, of these parameters will provide a useful indication of the potential of a molecular nanoparticle as siRNA transfecting vector.

Eosinophil cationic protein (ECP)/ribonuclease 3 is a member of the RNase A superfamily involved in inflammatory processes mediated by eosinophils. ECP is bactericidal, helminthotoxic, and cytotoxic to tracheal epithelium cells and to several mammalian cell lines although its RNase activity is low. We studied the thermal stability of ECP by fourth‐derivative UV absorbance spectra, circular dichroism, differential scanning calorimetry, and Fourier transform infrared spectroscopy. The T 1/2 values obtained with the different techniques were in very good agreement (T 1/2 ≈ 72°C), and the stability was maintained in the pH range between 5 and 7. The ECP calorimetric melting curve showed, in addition to the main transition, a pretransitional conformational change with a T 1/2 of 44°C. Both calorimetric transitions disappeared after successive re‐heatings, and the ratio ΔH versus ΔH vH of 2.2 indicated a significant deviation from the two‐state model. It was observed that the thermal unfo...

In the present work, we have thermodynamically characterized the thermally induced unfolding of 20 variants of bovine pancreatic ribonuclease A (RNase A) to experimentally describe the residues and the regions that are critical for the stability of the enzyme. The achieved results, complemented with previous studies by our group, allowed us to define the significance of the two hydrophobic nuclei present in the RNase A structure, as well as the contribution of the participating residues within each nucleus, to the global enzyme stability. We propose a structural model for the major and the minor hydrophobic nuclei of RNase A.

In this work we demonstrate that heat and pressure induce only slightly different energetic changes in the unfolded state of RNase A. Using pressure and temperature as denaturants on a significant number of variants, and by determining the free energy of unfolding at different temperatures, we estimated the stability of variants unable to complete the unfolding transition owing to the experimental conditions required for pressure experiments. The overall set of results allowed us to map the contributions to stability of the hydrophobic core residues of RNase A, with the positions most critical for stability being V54, V57, I106 and V108. We also show that the stability differences can be attributed to both hydrophobic interactions and packing density with an equivalent energetic magnitude. The main hydrophobic core of RNase A is tightly packed, as shown by the small-to-large and isosteric substitutions. In addition, we found that large changes in the number of methylene groups have ...

To investigate the characteristics of the postulated carboxy terminal chain-folding initiation site in bovine pancreatic ribonuclease A (RNase A) (residues 106-118), important in the early stages of the folding pathway, we have engineered by site-directed mutagenesis a set of 14 predominantly conservative hydrophobic variants of the protein. The stability of each variant has been compared by pressure and temperature-induced unfolding, monitored by fourth derivative UV absorbance spectroscopy. Apparently simple two-state, reversible unfolding transitions are observed, suggesting that the disruption of tertiary structure of each protein at high pressure or temperature is strongly cooperative. Within the limits of the technique, we are unable to detect significant differences between the two processes of denaturation. Both steady-state kinetic parameters for the enzyme reaction and UV CD spectra of each RNase A variant indicate that truncation of hydrophobic side chains in this region has, in general, little or no effect on the native structure and function of the enzyme. Furthermore, the decreases in free energy of unfolding upon pressure and thermal denaturation of all the variants, particularly those modified at residues 106 and 108, suggest that the hydrophobic residues and side chain packing interactions of this region play an important role in maintaining the conformational stability of RNase A. We also demonstrate the potential of Tyr115 replacement by Trp as a non-destabilizing fluorescence probe of conformational changes local to the region.

Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. Picornavirus IRES are organized in structural domains, in which the terminal stem–loops participate in functional RNA–protein interactions. However, the mechanistic role performed by the central domain during internal initiation has not been elucidated yet. Here we show that the foot-and-mouth-disease virus IRES contains a structural motif that serves in vitro as substrate for the Synechocystis sp. RNase P ribozyme, a structure-dependent endonuclease that participates in tRNA precursor processing. Recognition of the IRES substrate was dose dependent, required high magnesium concentration, and resulted in the formation of cleavage products with 5′ phosphate and 3′ hydroxyl ends. Mapping of the core recognition motif indicated that it overlapped with the apical region of the central domain. Two IRES constructs containing nucleotide substitutions...

The deafness-associated 7472insC mtDNA mutation was previously shown to decrease the steady-state level of tRNA Ser(UCN) post-transcriptionally. To identify the affected tRNA maturation step(s) we analysed the effects of the mutation on processing in vivo and in vitro. tRNA Ser(UCN) from cybrid cells homoplasmic for 7472insC contained a high frequency (>11%) of molecules misprocessed at one or both termini. In vitro assays using partially purified HeLa cell RNase P and mitochondrial tRNA 3 0 processing endonuclease (tRNase Z) confirmed that the efficiency of both 5 0 and 3 0 processing was impaired. A mutant precursor not already processed at the 5 0 end was poorly processed in vitro by tRNase Z. Misprocessing at the 3 0 end further impaired the efficiency and accuracy of 5 0 processing of the mutant substrate. The mutation thus appears to affect several distinct, but interdependent, RNA processing steps, with the predicted outcome dependent on the exact processing pathway operating in vivo.

Extracellular Mg 21 controls the activation of the Salmonella PhoP/PhoQ regulatory system. One of the adaptive responses driven by PhoP/PhoQ includes the transcriptional induction of mgtA and mgtCB, which encode two P-type Mg 21 transporters. Mg 21 also controls mgtA expression by a riboswitch located in its 5 0 -untranslated region (5 0 UTR). In this work, it was shown that the 5 0 UTR of both mgtA and mgtCB is responsible for a fine-tuned Mg 21 -dependent regulation of these genes. Evidence was also provided that the Mg 21 riboswitch targets the mgtA transcript for degradation by RNase E when cells are grown in high Mg 21 environments.

Escherichia coli RNase G is involved in the degradation of several mRNAs, including adhE and eno, which encode alcohol dehydrogenase and enolase respectively. Previous research indicates that the 5 0 untranslated region (5 0 -UTR) of adhE mRNA gives RNase G-dependency to lacZ mRNA when tagged at the 5 0 -end, but it has not been elucidated yet how RNase G recognizes adhE mRNA. Primer extension analysis revealed that RNase G cleaved a phosphodiester bond between À19A and À18C in the 5 0 -UTR (the A of the start codon was defined as þ1). Site-directed mutagenesis indicated that RNase G did not recognize the nucleotides at À19 and À18. Random deletion analysis indicated that the sequence from À145 to À125 was required for RNase G-dependent degradation. Secondary structure prediction and further site-directed deletion suggested that the stem-loop structure, with a bubble in the stem, is required for RNaseG-dependent degradation of adhE mRNA.

Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.

Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.

ABSTRACTCobalamin is an essential co-factor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea.Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen,M. tuberculosis, carries approximately 30 genes predicted to be involved inde novocobalamin biosynthesis.M. smegmatisalso encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase which catalyses the final reaction in methionine biosynthesis. In addition tometH,M. smegmatispossesses a cobalamin-independent methionine synthase,metE, suggesting that enzyme selection – MetH or MetE – is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controllingmetEexpression inM. tuberculosis. Here, we apply a targeted mass spectrometry-based approach to confirmde novocobalamin biosynthesis inM. smegmatisduring aerobic growthin vitro. We also demonstrate thatM. s...

Multidrug resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are non-viable. This essentiality supports it as a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two RNase HI enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knoc...

Abelmoschus esculentus (okra) is one of the polysaccharide rich crop plants. The polysaccharides interfere with nucleic acids and protein isolation thereby affecting the downstream molecular analysis. So, to understand the molecular systematics of okra, high quality DNA, RNA and proteins are essential. In this study we present a method for extracting genomic DNA, RNA and proteins from polysaccharide rich okra tissues. The conventional extraction procedures were integrated with purification treatments with pectinase, RNase and proteinase K, which improved the quality and quantity of DNA as well. Using SDS, additional washes with CIA and NaCl precipitation improved the RNA isolation both quantitatively and qualitatively. Finally, ammonium acetate mediated protein precipitation and re solubilization increased the quality of total protein extracts from the okra leaves. All of the methods above not only eliminated the impurities but also improved the quality and quantity of nucleic acids and proteins. Further, we subjected these samples to ver satile downstream molecular analyses such as restriction endonuclease digestion, RAPD, Southern, reverse transcription PCR and Western analysis and were proved to be successful.

The postantibiotic effect (PAE) values found for proteinase-defective (Lon−) Escherichia coli and RNase-defective E. coli exposed to antibiotics were reduced (31 to 60% and 35 to 50%, respectively) in comparison with the control (AB1157), and in the recA13 mutant these values were about 0.4 h with all drugs. Nalidixic acid, under anaerobic conditions, induced no PAE (0 to 0.1 h) in AB1157. A delay in regrowth (0.2 to 0.26 h) was noted with dnaA46(Ts), gyrA43(Ts), and gyrB41(Ts) mutants cultured for 2 h at 43°C. These findings suggest that when proteins and RNA are saved, the cell rapidly resumes the original growth rate.

External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. In this study, DNA-based EGS molecules were chemically synthesized to target the mRNA coding for the protease of human cytomegalovirus (HCMV). The EGS molecules efficiently directed human RNase P to cleave the target mRNA sequence in vitro . When EGSs were exogenously administered into HCMV-infected human foreskin fibroblasts, a reduction of about 80–90% in the expression level of the protease and a reduction of about 300-fold in HCMV growth were observed in the cells that were treated with a functional EGS, but not in cells that were not treated with the EGS or with a “disabled” EGS carrying nucleotide mutations that precluded RNase P recognition. Moreover, packaging of the viral DNA genome into the capsid was blocked in the cells treated with the functional EGS. These results indicate that HCMV ...

The aminoglycoside Geneticin (G418) is known to inhibit the cell culture proliferation, via virus-specific mechanisms, of two different virus genera from the Flaviviridae family. Here we try to determine whether Geneticin can selectively alter the switching of the 1-570 RNA region of the hepatitis C virus (HCV), and if so, whether or not this would inhibit viral growth. Two structure-dependent RNases known to specifically cleave HCV RNA were tested in the presence or absence of the drug. One was the Synechocystis sp. RNase P ribozyme, which cleaves the tRNA-like domain around the AUG start codon in high salt buffer conditions; the second was E. coli RNase III, which recognises a double-helical RNA switch element that changes the internal ribosome entry site (IRES) from a closed (C) conformation to an open (O) one. While the drug did not affect RNase P activity it did inhibit RNase III in the micromolar range. Kinetic studies indicated that the drug favours the switch from the C to O...

To get further insight into the factors involved in the maintenance of genome integrity we performed a screening of Saccharomyces cerevisiae deletion strains inducing hyperrecombination. We have identified trf4, a gene encoding a noncanonical polyA-polymerase involved in RNA surveillance, as a factor that prevents recombination between DNA repeats. We show that trf4D confers a transcription-associated recombination phenotype that is mediated by the nascent mRNA. In addition, trf4D also leads to an increase in the mutation frequency. Both genetic instability phenotypes can be suppressed by overexpression of RNase H and are exacerbated by overexpression of the human cytidine deaminase AID. These results suggest that in the absence of Trf4 R-loops accumulate co-transcriptionally increasing the recombination and mutation frequencies. Altogether our data indicate that Trf4 is necessary for both mRNA surveillance and maintenance of genome integrity, serving as a link between RNA and DNA metabolism in S. cerevisiae.

Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelialderived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo. Here we identified ribonuclease 6 (RNase 6) as the RNase A superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are upregulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14 þ monocytes and murine bone marrow-derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility.

Recent comparisons between plant and animal viruses reveal many common principles that underlie how all viruses express their genetic material, amplify their genomes, and link virion assembly with replication. Cauliflower mosaic virus (CaMV) is not infectious for human beings. Here, we show that CaMV transactivator/viroplasmin protein (TAV) shares sequence similarity with and behaves like the human ribonuclease H1 (RNase H1) in reducing DNA/RNA hybrids detected with S9.6 antibody in HEK293T cells. We showed that TAV is clearly expressed in the cytosol and in the nuclei of transiently transfected human cells, similar to its distribution in plants. TAV also showed remarkable cytotoxic effects in U251 human glioma cells in vitro. These characteristics pave the way for future analysis on the use of the plant virus protein TAV, as an alternative to human RNAse H1 during gene therapy in human cells.