Enhanced expression and purification of nucleotide-specific ribonucleases MC1 and Cusativin.

Combinations of ribonucleases (RNases) are commonly used to digest RNA into oligoribonucleotide fragments prior to liquid chromatography-mass spectrometry (LC-MS) analysis. The distribution of the RNase target sequences or nucleobase sites within an RNA molecule is critical for achieving a high mapping coverage. Cusativin and MC1 are nucleotide-specific endoribonucleases encoded in the cucumber and bitter melon genomes, respectively. Their high specificity for cytidine (Cusativin) and uridine (MC1) make them ideal molecular biology tools for RNA modification mapping. However, heterogenous recombinant expression of either enzyme has been challenging because of their high toxicity to expression hosts and the requirement of posttranslational modifications. Here, we present two highly efficient and time-saving protocols that overcome these hurdles and enhance the expression and purification of these RNases. We first purified MC1 and Cusativin from bacteria by expressing and shuttling both enzymes to the periplasm as MBP-fusion proteins in T7 Express lysY/IqE. coli strain at low temperature. The RNases were enriched using amylose affinity chromatography, followed by a subsequent purification via a C-terminal 6xHIS tag. This fast, two-step purification allows for the purification of highly active recombinant RNases significantly surpassing yields reported in previous studies. In addition, we expressed and purified a Cusativin-CBD fusion enzyme in P. pastoris using chitin magnetic beads. Both Cusativin variants exhibited a similar sequence preference, suggesting that neither posttranslational modifications nor the epitope-tags have a substantial effect on the sequence specificity of the enzyme.

Barnase encapsulation into submicron porous CaCO3 particles: studies of loading and enzyme activity.

The present study focuses on the immobilization of the bacterial ribonuclease barnase (Bn) into submicron porous calcium carbonate (CaCO3) particles. For encapsulation, we apply adsorption, freezing-induced loading and co-precipitation methods and study the effects of adsorption time, enzyme concentration and anionic polyelectrolytes on the encapsulation efficiency of Bn. We show that the use of negatively charged dextran sulfate (DS) and ribonucleic acid from yeast (RNA) increases the loading capacity (LC) of the enzyme on CaCO3 particles by about 3-fold as compared to the particles with Bn itself. The ribonuclease (RNase) activity of encapsulated enzyme depends on the LC of the particles and transformation of metastable vaterite to stable calcite, as studied by the assessment of enzyme activities in particles.

Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification.

BACKGROUND: The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar. RESULTS: The pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL- 1), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production. CONCLUSIONS: The current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.

Hypoxia Enhances the Expression of RNASET2 in Human Monocyte-Derived Dendritic Cells: Role of PI3K/AKT Pathway.

Hypoxia is a key component of the tumor microenvironment (TME) and promotes not only tumor growth and metastasis, but also negatively affects infiltrating immune cells by impairing host immunity. Dendritic cells (DCs) are the most potent antigen-presenting cells and their biology is weakened in the TME in many ways, including the modulation of their viability. RNASET2 belongs to the T2 family of extracellular ribonucleases and, besides its nuclease activity, it exerts many additional functions. Indeed, RNASET2 is involved in several human pathologies, including cancer, and it is functionally relevant in the TME. RNASET2 functions are not restricted to cancer cells and its expression could be relevant also in other cell types which are important players in the TME, including DCs. Therefore, this study aimed to unravel the effect of hypoxia (2% O2) on the expression of RNASET2 in DCs. Here, we showed that hypoxia enhanced the expression and secretion of RNASET2 in human monocyte-derived DCs. This paralleled the HIF-1α accumulation and HIF-dependent and -independent signaling, which are associated with DCs' survival/autophagy/apoptosis. RNASET2 expression, under hypoxia, was regulated by the PI3K/AKT pathway and was almost completely abolished by TLR4 ligand, LPS. Taken together, these results highlight how hypoxia- dependent and -independent pathways shape RNASET2 expression in DCs, with new perspectives on its implication for TME and, therefore, in anti-tumor immunity.

Group-2 innate lymphoid cell-dependent regulation of tissue neutrophil migration by alternatively activated macrophage-secreted Ear11.

Type-2 immunity is characterised by interleukin (IL)-4, IL-5 and IL-13, eosinophilia, mucus production, IgE, and alternatively activated macrophages (AAM). However, despite the lack of neutrophil chemoattractants such as CXCL1, neutrophils, a feature of type-1 immunity, are observed in type-2 responses. Consequently, alternative mechanisms must exist to ensure that neutrophils can contribute to type-2 immune reactions without escalation of deleterious inflammation. We now demonstrate that type-2 immune-associated neutrophil infiltration is regulated by the mouse RNase A homologue, eosinophil-associated ribonuclease 11 (Ear11), which is secreted by AAM downstream of IL-25-stimulated ILC2. Transgenic overexpression of Ear11 resulted in tissue neutrophilia, whereas Ear11-deficient mice have fewer resting tissue neutrophils, whilst other type-2 immune responses are not impaired. Notably, administration of recombinant mouse Ear11 increases neutrophil motility and recruitment. Thus, Ear11 helps maintain tissue neutrophils at homoeostasis and during type-2 reactions when chemokine-producing classically activated macrophages are infrequently elicited.

Semisynthesis of Human Ribonuclease-S.

Since its conception, the ribonuclease S complex (RNase S) has led to historic discoveries in protein chemistry, enzymology, and related fields. Derived by the proteolytic cleavage of a single peptide bond in bovine pancreatic ribonuclease (RNase A), RNase S serves as a convenient and reliable model system for incorporating unlimited functionality into an enzyme. Applications of the RNase S system in biomedicine and biotechnology have, however, been hindered by two shortcomings: (1) the bovine-derived enzyme could elicit an immune response in humans, and (2) the complex is susceptible to dissociation. Here, we have addressed both limitations in the first semisynthesis of an RNase S conjugate derived from human pancreatic ribonuclease and stabilized by a covalent interfragment cross-link. We anticipate that this strategy will enable unprecedented applications of the "RNase-S" system.

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita.

The edible mushroom Agrocybe aegerita produces a ribotoxin-like protein known as Ageritin. In this work, the gene encoding Ageritin was characterized by sequence analysis. It contains several typical features of fungal genes such as three short introns (60, 55 and 69 bp) located at the 5' region of the coding sequence and typical splice junctions. This sequence codes for a precursor of 156 amino acids (~17-kDa) containing an additional N-terminal peptide of 21 amino acid residues, absent in the purified toxin (135 amino acid residues; ~15-kDa). The presence of 17-kDa and 15-kDa forms was investigated by Western blot in specific parts of fruiting body and in mycelia of A. aegerita. Data show that the 15-kDa Ageritin is the only form retrieved in the fruiting body and the principal form in mycelium. The immunolocalization by confocal laser scanning microscopy and transmission electron microscopy proves that Ageritin has vacuolar localization in hyphae. Coupling these data with a bioinformatics approach, we suggest that the N-terminal peptide of Ageritin (not found in the purified toxin) is a new signal peptide in fungi involved in intracellular routing from endoplasmic reticulum to vacuole, necessary for self-defense of A. aegerita ribosomes from Ageritin toxicity.

The anti-inflammatory protein MCPIP1 inhibits the development of ccRCC by maintaining high levels of tumour suppressors.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. It is highly vascularized and largely resistant to traditional chemo- and radiotherapy. Decreases in tumour suppressors and low levels of the anti-inflammatory Monocyte Chemoattractant Protein-Induced Protein 1 (MCPIP1) play important roles in the development and progression of ccRCC. MCPIP1, also called Regnase-1, possesses endonuclease activity and degrades the mRNA of proinflammatory cytokines such as IL-6, IL-1ß, IL-12 and IL-2. We previously showed that the level of MCPIP1 decreases with ccRCC progression. In this study, we explored the role of MCPIP1 in regulating the levels of tumour suppressors. We found low levels of the suppressors PTEN, RECK and TIMP3 and high levels of MMPs in patients with ccRCC who had already been shown to have low MCPIP1 expression. We demonstrated that MCPIP1 regulates the expression levels of PTEN, RECK and TIMP3 in ccRCC cell lines as well as in vivo models of ccRCC. MCPIP1 overexpression increased the expression of tumour suppressors. Moreover, we observed that the RNase activity of MCPIP1 is responsible for the modulation of apoptosis and activation of prometastatic signalling pathways. Furthermore, we found a negative correlation between high levels of IL6, a direct target of MCPIP1 RNase activity, and TIMP3 in patients, indicating that MCPIP1 and TIMP3 might collectively cause the high levels of IL6 in ccRCC patients. Taken together, our results show the importance of MCPIP1 in regulating the level of tumour suppressors and, consequently, in ccRCC development and progression.

Expression and purification of the human tumor suppressor protein RNASET2 in CHO-S cells.

Members of the T2 extracellular ribonucleases family have long been reported as stress response proteins, often involved in host defence, in many different taxonomic groups. In particular, the human RNASET2 protein (hRNASET2) has been reported as an extracellular tumor suppressor protein, endowed with the ability to act as an "alarmin" signalling molecule following its expression and secretion in the tumor microenvironment by cancer cells and the subsequent recruitment and activation of cells belonging to the host innate immune system. Many in vitro and in vivo assays have been recently reported in support of the oncosuppressive role of hRNASET2: most of them relied on genetically engineered cell lines and the use of recombinant proteins from non-mammalian sources. In order to ensure a human-like glycosylation pattern, here we report for the first time the expression of recombinant hRNASET2 in the CHO-S cell line. We established a simple one-step chromatographic purification procedure that resulted in the production of 5 mg of endotoxin-free hRNASET2 per liter of culture, with a >95% purity degree. hRNASET2 expressed in CHO-S cells displayed a high degree of glycosylation homogeneity and a secondary structure content in agreement with that determined from the crystal structure. Indeed, recombinant hRNASET2 was active at both enzymatic and functional level, as stated by a biological activity assay. The availability of a pure, homogeneous recombinant human RNASET2 would provide a key tool to better investigate its non cell-autonomous roles in the context of cancer development and growth.

Preparation and Evaluation of Ribonuclease-Resistant Viral HIV RNA Standards Based on Armored RNA Technology

BAckground: The human immunodeficiency virus type 1 (HIV-1) is an infectious viral agent that gradually extinguishes the immune system, resulting in acquired immune deficiency syndrome (AIDS). The aim of this study was to construct an RNA-positive control based on armored (AR) RNA technology, using HIV-1 RNA as a model. Methods: The MS2 maturase, a coat protein gene (at positions 1765 to 1787) and HIV-1 pol gene were cloned into pET-32a plasmid. The prepared plasmid was transformed into Escherichia coli strain BL2 (DE3), and the expression of the construct was induced by 1 mM of isopropyl-L-thio-D-galactopyranoside (IPTG) at 37 °C for 16 h to obtain the fabricated AR RNA. The AR RNA was precipitated and purified using polyethylene glycol and Sephacryl S-200 chromatography. Results: The stability of AR RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy and gel agarose electrophoresis. Tenfold serial dilution of AR RNA from 101 to 105 was prepared. Real-time PCR assays had a range of detection between 101 and 105. In addition, R2 value was 0.998, and the slope of the standard curve was -3.33. Conclusion: Prepared AR RNA, as a positive control, could be used as a basis for launching an in-house HIV-1 virus assay and other infectious agents. It can be readily available to laboratories and HIV research centers. The AR RNA is non-infectious and highly resistant to ribonuclease enzyme and can reduce the risk of infection in the clinical laboratory.

RNase 7 Strongly Promotes TLR9-Mediated DNA Sensing by Human Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) were described to accumulate in the skin of patients with psoriasis and to be recruited into the dermis upon allergen challenge in atopic dermatitis. Activation of pDCs in the skin has been identified as an important initiator of psoriasis development. Ribonuclease (RNase) 7 is one of the major antimicrobial peptides secreted by keratinocytes and is expressed in significantly higher amounts in lesional skin of patients with atopic dermatitis or psoriasis than in healthy individuals. The skin-derived antimicrobial peptides human ß-defensin 2 and LL-37 indirectly stimulate the activity of skin pDCs, but to our knowledge, an immunomodulatory potential of RNase 7 has not yet been reported. We show here that RNase 7 enables human pDCs to recognize self-DNA and promotes their rapid sensing of bacterial DNA. This very fast innate immune response was sufficient to up-regulate the expression of several antiviral IFN-stimulated genes in human peripheral blood mononuclear cells and to inhibit an infection of primary human keratinocytes with herpes simplex virus 1. RNase 7 was a markedly stronger trigger for IFN-α expression in human pDCs than the other antimicrobial peptides. Our data indicate that RNase 7 exhibits potent immunomodulatory functions and supports the efficient recognition of microbial infections by human skin-infiltrating pDCs.

p38 but not p53 is responsible for UVA-induced MCPIP1 expression.

MCPIP1 (Monocyte Chemotactic Protein-1 Induced Protein) is an important regulator of inflammation and cell apoptosis, but its role in UVA-induced stress response in the epidermis has never been studied. We have found that moderate apoptosis-inducing dose of UVA (27J/cm2) increases the level of MCPIP1 expression in HaCaT cells and normal human keratinocytes (NHEK) within 6-9h after the treatment. MCPIP1 upregulation was dependent on the induction of p38, but not p53, as demonstrated by using p38 inhibitor SB203580 and p53 inducer RG7388, respectively. This increase was also blocked by antioxidants (α-tocopherol and ascorbic acid), suggesting the involvement of MCPIP1 in UVA-induced oxidative stress response. Si-RNA-mediated down-regulation of MCPIP1 expression in HaCaT cells resulted in increased sensitivity to UVA-induced DNA damage and apoptosis. This was accompanied by decreased phosphorylation of p53 and p38 in MCPIP1-silenced cells following UVA irradiation. The activation of p38 in response to low doses of ultraviolet radiation was postulated to be protective for p53-inactive cells. Therefore, MCPIP1 may favor the survival of p53-defective HaCaT cells by sustaining the activation of p38. This creates a loop of mutual positive regulation between p38 and MCPIP1 protein in HaCaT cells, providing the protection against the consequences of UVA irradiation.

Histone deacetylase inhibitor vorinostat (SAHA, MK0683) perturb miR-9-MCPIP1 axis to block IL-1ß-induced IL-6 expression in human OA chondrocytes.

AIM OF THE STUDY: High levels of IL-6 are believed to contribute to osteoarthritis (OA) pathogenesis. The expression of IL-6 is regulated post-transcriptionally by the miR-9-MCPIP-1 axis in chondrocytes. Vorinostat (SAHA) inhibits the IL-6 expression in OA chondrocytes. We investigated whether SAHA suppresses the expression of IL-6 by perturbing the miR-9-MCPIP1 axis in OA chondrocytes under pathological conditions. MATERIALS AND METHODS: OA chondrocytes were isolated by enzymatic digestion and treated with IL-1ß in the absence or presence of SAHA. Genes and protein expression levels were determined by TaqMan assays and Western blotting, respectively. Secreted IL-6 was quantified by enzyme linked immunosorbent assay (ELISA). MCPIP1 promoter deletion mutants were generated by polymerase chain reaction (PCR). Promoter recruitment of transcription factors was determined by ChIP. Nuclear run-on was employed to measure the ongoing transcription. siRNA-mediated knockdown of the CEBPα expression was employed for loss of function studies. RESULTS: Expression of MCPIP1 was high in SAHA treated OA chondrocytes but expression of IL-6 mRNAs and secreted IL-6 were reduced by ~70%. SAHA suppressed the expression of miR-9 but enhanced the activity of the MCPIP1 promoter localized to a 156bp region which also harbors the binding site for CEBPα. Treatment with SAHA enhanced the recruitment of CEBPα to the MCPIP1 promoter. Ectopically expressed CEBPα enhanced the promoter activity and the expression of MCPIP1 while siRNA-mediated knockdown of CEBPα inhibited the expression of MCPIP1. CONCLUSIONS: Taken together our data indicate that SAHA-mediated suppression of the IL-6 expression is achieved through increased recruitment of CEBPα to the MCPIP1 promoter and by relieving the miR-9-mediated inhibition of MCPIP1 expression in OA chondrocytes.

A Recessive Pollination Control System for Wheat Based on Intein-Mediated Protein Splicing.

A transgene-expression system for wheat that relies on the complementation of inactive precursor protein fragments through a split-intein system is described. The N- and C-terminal fragments of a barnase gene from Bacillus amyloliquifaciens were fused to intein sequences from Synechocystis sp. and transformed into wheat plants. Upon translation, both barnase fragments are assembled by an autocatalytic intein-mediated trans-splicing reaction, thus forming a cytotoxic enzyme. This chapter focuses on the use of introns and flexible polypeptide linkers to foster the expression of a split-barnase expression system in plants. The methods and protocols that were employed with the objective to test the effects of such genetic elements on transgene expression and to find the optimal design of expression vectors for use in wheat are provided. Split-inteins can be used to form an agriculturally important trait (male sterility) in wheat plants. The use of this principle for the production of hybrid wheat seed is described. The suggested toolbox will hopefully be a valuable contribution to future optimization strategies in this commercially important crop.

Intact NYN/PIN-Like Domain is Crucial for the Degradation of Inflammation-Related Transcripts by ZC3H12D.

ZC3H12D belongs to a recently discovered family of proteins containing four members of which the most studied and best described is the RNase ZC3H12A (MCPIP1/Regnase-1). ZC3H12A is a crucial negative regulator of inflammation. It accelerates the turnover of transcripts of a spectrum of proinflammatory cytokines, as well as its own mRNA. The biological role of ZC3H12D is less clear, although it was shown that this member of ZC3H12 family is also involved in the regulation of inflammation. Here, we show that ZC3H12A and ZC3H12D recognize a set of common target mRNAs encoding proteins that play important roles in the course of the inflammation. Similarly to ZC3H12A, ZC3H12D participates in the 3'UTR-dependent regulation of the turnover of mRNAs encoding interleukin-6 (IL-6), tumor necrosis factor (TNF), and immediate early response 3 gene (IER3). The ZC3H12A mRNA is also among the identified ZC3H12D targets. Using the combination of immunofluorescence with single molecule RNA fluorescence in situ hybridization (smRNA FISH) we have shown that ZC3H12D protein interacts with the ZC3H12A transcript. The direct binding of these two molecules in vivo was further confirmed by RNA immunoprecipitation. Simultaneously, overexpression of ZC3H12D increases the turnover rate of transcripts containing ZC3H12A 3'UTR. Using reporter gene assays we have confirmed that the Asp95 residue present in the NYN/PIN-like domain is crucial for ZC3H12D biological activity. We have also revealed that ZC3H12D recognizes the same structural elements present in the 3'UTRs of the investigated transcripts, as ZC3H12A. J. Cell. Biochem. 118: 487-498, 2017. © 2016 Wiley Periodicals, Inc.

Expression Patterns and Functional Novelty of Ribonuclease 1 in Herbivorous Megalobrama amblycephala.

Ribonuclease 1 (RNase1) is an important digestive enzyme that has been used to study the molecular evolutionary and plant-feeding adaptation of mammals. However, the expression patterns and potential biological function of RNase1 in herbivorous fish is not known. Here, we identified RNase1 from five fish species and illuminated the functional diversification and expression of RNase1 in herbivorous Megalobrama amblycephala. The five identified fish RNase1 genes all have the signature motifs of the RNase A superfamily. No expression of Ma-RNase1 was detected in early developmental stages but a weak expression was detected at 120 and 144 hours post-fertilization (hpf). Ma-RNase1 was only expressed in the liver and heart of one-year-old fish but strongly expressed in the liver, spleen, gut, kidney and testis of two-year-old fish. Moreover, the immunostaining localized RNase1 production to multiple tissues of two-year-old fish. A biological functional analysis of the recombinant protein demonstrated that M. amblycephala RNase1 had a relatively strong ribonuclease activity at its optimal pH 6.1, which is consistent with the pH of its intestinal microenvironment. Collectively, these results clearly show that Ma-RNase1 protein has ribonuclease activity and the expression patterns of Ma-RNase1 are dramatically different in one year and two-year-old fish, suggesting the functional differentiation during fish growing.

Cell-free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just-add-water cell-free system.

Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult-to-express proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize cell-free protein synthesis. Cell-free systems offer a compelling alternative to in vivo production, due to their open and malleable reaction environments. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. This lyophilized system remains viable after being stored above freezing for up to one year. The beneficial features of these cell-free systems make them compelling candidates for future biotherapeutic screening and production.

Differential induction of innate defense antimicrobial peptides in primary nasal epithelial cells upon stimulation with inflammatory cytokines, Th17 cytokines or bacterial conditioned medium from Staphylococcus aureus isolates.

To date it is incompletely understood why half of the human population is intrinsically resistant to Staphylococcus aureus colonization whereas the other half is intermittently or permanently colonized. Nasal colonization represents the primary niche for S. aureus. We therefore investigated whether primary nasal epithelial cells (HNEC) express antimicrobial peptides (AMPs) upon stimulation by inflammatory cytokines or bacterial conditioned medium (BCM) of different colonizing and invasive staphylococci. Stimulation with classical cytokines (IL-1ß, TNF-α, IFN-γ) potently induced hBD-3 and RNase7 in HNEC. Th17 cytokines (IL-17A, IL-17F, IL-22) yielded comparably weak hBD-3 and RNase7 induction and no synergistic effects with classical cytokines. BCM of S. aureus and Staphylococcus epidermidis isolates moderately induced hBD3 and RNase7 mRNA expression without significant differences when comparing colonizing vs. invasive isolates. Our results indicate that HNEC contribute to the innate defense by secretion of an AMP-containing chemical defense shield along the nasal mucosa i.e. within the primary colonization niche of S. aureus. Further studies are needed to investigate whether a deficient AMP expression in the nasal mucosa may be related to different S. aureus carrier states. AMPs or AMP-inducing agents may be promising candidates for future topical decolonization regimens that aim to prevent invasive S. aureus infections.

Decay-Initiating Endoribonucleolytic Cleavage by RNase Y Is Kept under Tight Control via Sequence Preference and Sub-cellular Localisation.

Bacteria depend on efficient RNA turnover, both during homeostasis and when rapidly altering gene expression in response to changes. Nevertheless, remarkably few details are known about the rate-limiting steps in targeting and decay of RNA. The membrane-anchored endoribonuclease RNase Y is a virulence factor in Gram-positive pathogens. We have obtained a global picture of Staphylococcus aureus RNase Y sequence specificity using RNA-seq and the novel transcriptome-wide EMOTE method. Ninety-nine endoribonucleolytic sites produced in vivo were precisely mapped, notably inside six out of seven genes whose half-lives increase the most in an RNase Y deletion mutant, and additionally in three separate transcripts encoding degradation ribonucleases, including RNase Y itself, suggesting a regulatory network. We show that RNase Y is required to initiate the major degradation pathway of about a hundred transcripts that are inaccessible to other ribonucleases, but is prevented from promiscuous activity by membrane confinement and sequence preference for guanosines.

Microbial ribonucleases (RNases): production and application potential.

Ribonuclease (RNase) is hydrolytic enzyme that catalyzes the cleavage of phosphodiester bonds in RNA. RNases play an important role in the metabolism of cellular RNAs, such as mRNA and rRNA or tRNA maturation. Besides their cellular roles, RNases possess biological activity, cell stimulating properties, cytotoxicity and genotoxicity. Cytotoxic effect of particular microbial RNases was comparable to that of animal derived counterparts. In this respect, microbial RNases have a therapeutic potential as anti-tumor drugs. The significant development of DNA vaccines and the progress of gene therapy trials increased the need for RNases in downstream processes. In addition, RNases are used in different fields, such as food industry for single cell protein preparations, and in some molecular biological studies for the synthesis of specific nucleotides, identifying RNA metabolism and the relationship between protein structure and function. In some cases, the use of bovine or other animal-derived RNases have increased the difficulties due to the safety and regulatory issues. Microbial RNases have promising potential mainly for pharmaceutical purposes as well as downstream processing. Therefore, an effort has been given to determination of optimum fermentation conditions to maximize RNase production from different bacterial and fungal producers. Also immobilization or strain development experiments have been carried out.