Function Of Ribonuclease Research Articles

The role of the 5’ sensing function of ribonuclease E in cyanobacteria

ABSTRACT RNA degradation is critical for synchronising gene expression with changing conditions in prokaryotic and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y for 5’-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5’ sensing, contrasting to the alternative ‘direct entry’ mode, which is independent of monophosphorylated 5’ ends. Cyanobacteria, such as Synechocystis sp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologues. Here, we constructed a Synechocystis strain lacking the 5’ sensing function of RNase E and mapped on a transcriptome-wide level 283 5’-sensing-dependent cleavage sites. These included so far unknown targets such as mRNAs encoding proteins related to energy metabolism and carbon fixation. The 5’ sensing function of cyanobacterial RNase E is important for the maturation of rRNA and several tRNAs, including tRNAGlu UUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and in most prokaryotes. Furthermore, we found that increased RNase activities lead to a higher copy number of the major Synechocystis plasmids pSYSA and pSYSM. These results provide a first step towards understanding the importance of the different target mechanisms of RNase E outside Escherichia coli.

Advances in application of CRISPR-Cas13a system.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins serve as an adaptive immune system that safeguards prokaryotes and some of the viruses that infect prokaryotes from foreign nucleic acids (such as viruses and plasmids). The genomes of the majority of archaea and about half of all bacteria contain various CRISPR-Cas systems. CRISPR-Cas systems depend on CRISPR RNAs (crRNAs). They act as a navigation system to specifically cut and destroy foreign nucleic acids by recognizing invading foreign nucleic acids and binding Cas proteins. In this review, we provide a brief overview of the evolution and classification of the CRISPR-Cas system, focusing on the functions and applications of the CRISPR-Cas13a system. We describe the CRISPR-Cas13a system and discuss its RNA-directed ribonuclease function. Meanwhile, we briefly introduce the mechanism of action of the CRISPR-Cas13a system and summarize the applications of the CRISPR-Cas13a system in pathogen detection, eukaryotes, agriculture, biosensors, and human gene therapy. We are right understanding of CRISPR-Cas13a has been broadened, and the CRISPR-Cas13a system will be useful for developing new RNA targeting tools. Therefore, understanding the basic details of the structure, function, and biological characterization of CRISPR-Cas13a effector proteins is critical for optimizing RNA targeting tools.

Domain swapping of the C-terminal helix promotes the dimerization of a novel ribonuclease protein from Mycobacterium tuberculosis.

Polyketide metabolism-associated proteins in Mycobacterium tuberculosis play an essential role in the survival of the bacterium, which makes them potential drug targets for the treatment of tuberculosis (TB). The novel ribonuclease protein Rv1546 is predicted to be a member of the steroidogenic acute regulatory protein-related lipid-transfer (START) domain superfamily, which comprises bacterial polyketide aromatase/cyclases (ARO/CYCs). Here, we determined the crystal structure of Rv1546 in a V-shaped dimer. The Rv1546 monomer consists of four α-helices and seven antiparallel β-strands. Interestingly, in the dimeric state, Rv1546 forms a helix-grip fold, which is present in START domain proteins, via three-dimensional domain swapping. Structural analysis revealed that the conformational change of the C-terminal α-helix of Rv1546 might contribute to the unique dimer structure. Site-directed mutagenesis followed by in vitro ribonuclease activity assays was performed to identify catalytic sites of the protein. This experiment suggested that surface residues R63, K84, K88, and R113 are important in the ribonuclease function of Rv1546. In summary, this study presents the structural and functional characterization of Rv1546 and supplies new perspectives for exploiting Rv1546 as a novel drug target for TB treatment.

A secreted ribonuclease effector from Verticillium dahliae localizes in the plant nucleus to modulate host immunity.

The arms race between fungal pathogens and plant hosts involves recognition of fungal effectors to induce host immunity. Although various fungal effectors have been identified, the effector functions of ribonucleases are largely unknown. Herein, we identified a ribonuclease secreted by Verticillium dahliae (VdRTX1) that translocates into the plant nucleus to modulate immunity. The activity of VdRTX1 causes hypersensitive response (HR)‐related cell death in Nicotiana benthamiana and cotton. VdRTX1 possesses a signal peptide but is unlikely to be an apoplastic effector because its nuclear localization in the plant is necessary for cell death induction. Knockout of VdRTX1 significantly enhanced V. dahliae virulence on tobacco while V. dahliae employs the known suppressor VdCBM1 to escape the immunity induced by VdRTX1. VdRTX1 homologs are widely distributed in fungi but transient expression of 24 homologs from other fungi did not yield cell death induction, suggesting that this function is specific to the VdRTX1 in V. dahliae. Expression of site‐directed mutants of VdRTX1 in N. benthamiana leaves revealed conserved ligand‐binding sites that are important for VdRTX1 function in inducing cell death. Thus, VdRTX1 functions as a unique HR‐inducing effector in V. dahliae that contributes to the activation of plant immunity.

Ceratocystis cacaofunesta differentially modulates the proteome in xylem-enriched tissue of cocoa genotypes with contrasting resistance to Ceratocystis wilt.

Decreased accumulation of polyphenol oxidase, H2O2 accumulation, effective regulation of programmed cell death, and a protein predicted as allergenic can play key roles in cacao defense against Ceratocystis cacaofunesta. Ceratocystis wilt, caused by the fungus Ceratocystis cacaofunesta, has destroyed millions of Theobroma cacao trees in several countries of the Americas. Through proteomics, systems biology, and enzymatic analyses of infected stems, it was possible to infer mechanisms used by resistant (TSH1188) and susceptible (CCN51) cacao genotypes during infection. Protein extraction from xylem-enriched tissue of stems inoculated with the fungus and their controls 1day after inoculation was carried out, followed by separation through two-dimensional gel electrophoresis and identification by mass spectrometry. Enzyme activity was determined at 1, 3, 7 and 15days after inoculation. A total of 50 differentially accumulated distinct proteins were identified in the treatments of both genotypes and were classified into 10 different categories. An interaction network between homologous proteins from Arabidospsis thaliana was generated for each genotype, using the STRING database and Cytoscape software. Primary metabolism processes were apparently repressed in both genotypes. The resistance factors suggested for genotype TSH1188 were: H2O2 accumulation, effective regulation of programmed cell death, production of phytoalexins derived from tryptophan and furanocoumarins, and participation of a predicted allergenic protein with probable ribonuclease function inhibiting the germination and propagation of the fungus. In the susceptible genotype, it is possible that its recognition and signaling mechanism through proteins from the SEC14 family is easily overcome by the pathogen. Our results will help to better understand the interaction between cacao and one of its most aggressive pathogens, to create disease control strategies.

CircRNA RPPH1 Facilitates the Aggravation of Breast Cancer Development by Regulating miR-542-3p/ARHGAP1 Pathway.

Background: Circular RNAs (circRNAs) have important roles in human malignancies, including breast cancer (BC). In this study, we explored the function of circRNA ribonuclease P RNA component H1 (circ_RPPH1) in BC development and clarify the mechanistic pathway. Materials and Methods: Expression of circ_RPPH1, microRNA-542-3p (miR-542-3p), and Rho GTPase-activating protein 1 (ARHGAP1) in BC tissues and cells was determined by quantitative real-time polymerase chain reaction or Western blot assay. The stability of circ_RPPH1 was confirmed by RNase R and actinomycin D treatment. Cell viability and colony formation ability were measured by methyl thiazolyl tetrazolium (MTT) assay and colony formation assay, respectively. Western blot analysis was also used to detect proliferation biomarker (Ki67) and epithelial-mesenchymal transition (EMT) biomarkers (E-cadherin, N-cadherin, and vimentin). Flow cytometry and Transwell assays were performed to monitor cell apoptosis, migration, and invasion. The binding potency between miR-542-3p and circ_RPPH1 or ARHGAP1 was validated by dual-luciferase reporter assay. Functional role of circ_RPPH1 in vivo was investigated by xenograft tumor reporter assay. Results: Upregulation of circ_RPPH1 and ARHGAP1, and downregulation of miR-542-3p were detected in BC tissues and cells. circ_RPPH1 knockdown or miR-542-3p introduction inhibited BC cell proliferation and metastasis, while promoted apoptosis in vitro. circ_RPPH1 sponged miR-542-3p to upregulate ARHGAP1 expression, thereby affecting BC progression. Moreover, depletion of circ_RPPH1 suppressed tumor growth in vivo. Conclusions: circ_RPPH1 contributed to BC tumorigenesis by sponging miR-542-3p and upregulating ARHGAP1, affording a novel mechanistic pathway in BC development.

Expression and function of human ribonuclease 4 in the kidney and urinary tract.

Antimicrobial peptides are essential host defense mechanisms that prevent urinary tract infections. Recent studies have demonstrated that peptides in the ribonuclease A superfamily have antimicrobial activity against uropathogens and protect the urinary tract from uropathogenic Escherichia coli (UPEC). Little is known about the antibacterial function or expression of ribonuclease 4 (RNase 4) in the human urinary tract. Here, we show that full-length recombinant RNase 4 peptide and synthetic amino-terminal RNase 4 peptide fragment have antibacterial activity against UPEC and multidrug-resistant (MDR)-UPEC. RNASE4 transcript expression was detected in human kidney and bladder tissue using quantitative real-time PCR. Immunostaining or in situ hybridization localized RNase 4 expression to proximal tubules, principal and intercalated cells in the kidney's collecting duct, and the bladder urothelium. Urinary RNase 4 concentrations were quantified in healthy controls and females with a history of urinary tract infection. Compared with controls, urinary RNase 4 concentrations were significantly lower in females with a history of urinary tract infection. When RNase 4 was neutralized in human urine or silenced in vitro using siRNA, urinary UPEC replication or attachment to and invasion of urothelial and kidney medullary cells increased. These data show that RNase 4 has antibacterial activity against UPEC, is expressed in the human urinary tract, and can contribute to host defense against urinary tract infections.NEW & NOTEWORTHY Ribonuclease 4 (RNase 4) is a newly identified host defense peptide in the human kidney and bladder. RNase 4 kills uropathogenic Escherichia coli (UPEC) and multidrug-resistant UPEC. RNase 4 prevents invasive UPEC infection and suppressed RNase 4 expression may be a risk factor for more severe or recurrent urinary tract infection.

Writing a review on trapping RNase substrate complexes for structural studies as an undergraduate researcher

We conducted a major survey of ribonuclease (RNase) substrate complex structures to identify strategies used to inhibit cleavage of the RNA. This review is intended to serve as a guide for structural biologists interested in discovering mechanisms and functions of ribonucleases through structural studies. We searched the Protein Data Bank for RNase structures which include both RNase and nucleic acid substrate and vetted the results for structures in which the substrate remained intact. Then we organized the structures by category based on the method used for inhibition. Each of the methods include their owns pros and cons and often it is advantageous to use a mix of different methods to achieve inhibition. In addition to surveying current strategies, we identify some ideas for future directions in the field based on what we have learned.

Genetic Insight into the Domain Structure and Functions of Dicer-Type Ribonucleases.

Ribonuclease Dicer belongs to the family of RNase III endoribonucleases, the enzymes that specifically hydrolyze phosphodiester bonds found in double-stranded regions of RNAs. Dicer enzymes are mostly known for their essential role in the biogenesis of small regulatory RNAs. A typical Dicer-type RNase consists of a helicase domain, a domain of unknown function (DUF283), a PAZ (Piwi-Argonaute-Zwille) domain, two RNase III domains, and a double-stranded RNA binding domain; however, the domain composition of Dicers varies among species. Dicer and its homologues developed only in eukaryotes; nevertheless, the two enzymatic domains of Dicer, helicase and RNase III, display high sequence similarity to their prokaryotic orthologs. Evolutionary studies indicate that a combination of the helicase and RNase III domains in a single protein is a eukaryotic signature and is supposed to be one of the critical events that triggered the consolidation of the eukaryotic RNA interference. In this review, we provide the genetic insight into the domain organization and structure of Dicer proteins found in vertebrate and invertebrate animals, plants and fungi. We also discuss, in the context of the individual domains, domain deletion variants and partner proteins, a variety of Dicers’ functions not only related to small RNA biogenesis pathways.

Identification of a perchloric acid-soluble protein (PSP)-like ribonuclease from Trichomonas vaginalis

A perchloric acid-soluble protein (PSP), named here tv-psp1, was identified in Trichomonas vaginalis. It is expressed under normal culture conditions according to expressed sequence tag (EST) analysis. On the other hand, Tv-PSP1 protein was identified by mass spectrometry with a 40% of identity to human PSP (p14.1). Polyclonal antibodies against recombinant Tv-PSP1 (rTv-PSP1) recognized a single band at 13.5kDa in total protein parasite extract by SDS-PAGE and a high molecular weight band analyzed by native PAGE. Structural analysis of Tv-PSP1, using dynamic light scattering, size exclusion chromatography, and circular dichroism spectroscopy, showed a trimeric structure stable at 7M urea with 38% α-helix and 14% β-sheet in solution and a molecular weight of 40.5kD. Tv-PSP1 models were used to perform dynamic simulations over 100ns suggesting a stable homotrimeric structure. Tv-PSP1 was located in the nucleus, cytoplasm, and hydrogenosomes of T. vaginalis, and the in silico analysis by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) showed interactions with RNA binding proteins. The preliminary results of RNA degradation analysis with the recombinant Tv-PSP1 showed RNA partial deterioration suggesting a possible putative ribonuclease function.

Regulation of cytoplasmic RNA stability: Lessons from Drosophila.

The process of RNA degradation is a critical level of regulation contributing to the control of gene expression. In the last two decades a number of studies have shown the specific and targeted nature of RNA decay and its importance in maintaining homeostasis. The key players within the pathways of RNA decay are well conserved with their mutation or disruption resulting in distinct phenotypes as well as human disease. Model organisms including Drosophila melanogaster have played a substantial role in elucidating the mechanisms conferring control over RNA stability. A particular advantage of this model organism is that the functions of ribonucleases can be assessed in the context of natural cells within tissues in addition to individual immortalized cells in culture. Drosophila RNA stability research has demonstrated how the cytoplasmic decay machines, such as the exosome, Dis3L2 and Xrn1, are responsible for regulating specific processes including apoptosis, proliferation, wound healing and fertility. The work discussed here has begun to identify specific mRNA transcripts that appear sensitive to specific decay pathways representing mechanisms through which the ribonucleases control mRNA stability. Drosophila research has also contributed to our knowledge of how specific RNAs are targeted to the ribonucleases including AU rich elements, miRNA targeting and 3' tailing. Increased understanding of these mechanisms is critical to elucidating the control elicited by the cytoplasmic ribonucleases which is relevant to human disease. This article is categorized under: RNA in Disease and Development > RNA in Development RNA Turnover and Surveillance > Regulation of RNA Stability RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms.

Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle.

Modifying RNA through either splicing or editing is a fundamental biological process for creating protein diversity from the same genetic code. Developing novel chemical biology tools for RNA editing has potential to transiently edit genes and to provide a better understanding of RNA biochemistry. Current techniques used to modify RNA include the use of ribozymes, adenosine deaminase, and tRNA endonucleases. Herein, we report a nanozyme that is capable of splicing virtually any RNA stem-loop. This nanozyme is comprised of a gold nanoparticle functionalized with three enzymes: two catalytic DNA strands with ribonuclease function and an RNA ligase. The nanozyme cleaves and then ligates RNA targets, performing a splicing reaction that is akin to the function of the spliceosome. Our results show that the three-enzyme reaction can remove a 19 nt segment from a 67 nt RNA loop with up to 66% efficiency. The complete nanozyme can perform the same splice reaction at 10% efficiency. These splicing nanozymes represent a new promising approach for gene manipulation that has potential for applications in living cells.

Biochemical characterization of Type II Toxin‐Antitoxin module from Pseudomonas aeruginosa

To date numerous putative Toxin‐Antitoxin (TA) loci have been reported, and these consist of a dicistronic operon encoding two genes; one for antibacterial toxic protein and another for antitoxin which neutralizes its specific toxin (Leplae et al., 2011). TA systems have been reported to play numerous physiological roles, including formation of persister cells (Maisonneuve et al., 2011 & Li et al., 2016), stress resistance (Christensen et al., 2001), protection from bacteriophages (Koga et al., 2011) and regulation of biofilm formation (Wang et al., 2011). Further, overexpression of a Hig‐type toxin affects the virulence factor production that is crucial for modulating Pseudomonas infection (Wood TL, Wood TK. 2016).The goal of this study is to characterize a highly conserved member of the ParE/RelE superfamily from Pseudomonas aeruginosa. Toxin and antitoxin genes were cloned into the pHERD shuttle expression vector for propagation in both E. coli and P. aeruginosa. Heterologous over‐expression of the toxin in E.coli promoted a filamentous phenotype and decreased viability based on enumerating the colony forming unit, while overexpression of the antitoxin did not affect the cell count or the phenotype. We developed purification strategies to purify RelE toxin in the absence of any binding partners, as well as the RelBE protein complex. Gel filtration purification revealed mixtures of dimers of both proteins in complex (RelB2E2) and also fractions with molecular weight matching the free RelE monomer mass. Upon examining the sequence of this RelE toxin, the longer helices associated with a ParE gyrase inhibiting toxin were evident. Experimental testing has clearly demonstrated the ability of this toxin to inhibit DNA gyrase, and studies are underway to evaluate potential ribonuclease function as expected from its annotation. However, based on the longer helices, the region important for specificity for cellular targets, we expect that this RelE toxin is instead a ParE toxin. Additionally we have been successful in obtaining crystals of RelE and evaluated their diffraction properties at the OU Macromolecular crystallography facility, which yielded weak reflections to 4.5 Å. Initial crystallization condition for the RelBE complex have been identified and current efforts are focused on optimizing these for analysis by crystallography. The determination of the structure of this TA module will be the first from P. aeruginosa. Our overall goal of combining structural features and functionality of this TA module will facilitate understanding its role in bacterial physiology, a promising approach for combating this multidrug resistant pathogen.Support or Funding InformationThis work was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health.

Universal RNA-degrading enzymes in Archaea: Prevalence, activities and functions of β-CASP ribonucleases.

β-CASP ribonucleases are widespread in all three domains of life. They catalyse both 5'-3' exoribonucleolytic RNA trimming and/or endoribonucleolytic RNA cleavage using a unique active site coordinated by two zinc ions. These fascinating enzymes have a key role in 3' end processing in Eukarya and in RNA decay and ribosomal RNA maturation in Bacteria. The recent recognition of β-CASP ribonucleases as major players in Archaea is an important contribution towards identifying RNA-degrading enzymes in the third domain of life. Three β-CASP orthologous groups, aCPSF1, aCPSF2, aCPSF1b, are closely related to the eukaryal CPSF73 termination factor and one, aRNase J, is ortholog of the bacterial RNase J. The endo- and 5'-3'exoribonucleolytic activities carried by archaeal β-CASP enzymes are strictly conserved throughout archaeal phylogeny suggesting essential roles in maturation and/or degradation of RNA. The recent progress in understanding the prevalence, activities and functions of archaeal β-CASP ribonucleases is the focus of this review. The current status of our understanding of RNA processing pathways in Archaea is covered in light of this new knowledge on β-CASP ribonucleases.

Inhibition of hepatitis B virus replication by ligand-mediated activation of RNase L

RNase L is a cellular endoribonuclease that is activated by 2′,5′-linked oligoadenylates (2–5A), which are unique and specific ligands synthesized by a family of interferon-inducible, dsRNA-activated enzymes named oligoadenylate synthetases. In the typical antiviral pathway, activated RNase L degrades viral and cellular RNAs, thus limiting viral replication and spread. Although the antiviral activity of RNase L has been demonstrated for several RNA viruses, there is little evidence regarding its role against DNA viruses. In the present study, the potential antiviral activity of RNase L against hepatitis B virus (HBV) was explored utilizing the recently reported infection protocol based on human hepatoma HepG2 cells stably complemented with the virus entry factor NTCP. Viral replication and expression in this cell type was markedly inhibited by poly(I:C)- or 2–5A-mediated activation of RNase L; however, the inhibition was significantly reversed by RNase L knockdown. Further analysis in HBV1.2-transfected Huh-7 hepatoma cells indicated that the antiviral activity of RNase L depends on its ribonuclease function. We also provide evidence for the specific roles of OAS family members in this process. These results suggest that HBV replication can be regulated through interferon-mediated RNA decay pathways and that activation of these host antiviral factors may represent a novel therapeutic strategy for HBV infection.

S-like ribonuclease gene expression in carnivorous plants

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.

Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

Plasmacytoid dendritic cells (pDC) have been shown to efficiently sense HCV- or HIV-infected cells, using a virion-free pathway. Here, we demonstrate for classical swine fever virus, a member of the Flaviviridae, that this process is much more efficient in terms of interferon-alpha induction when compared to direct stimulation by virus particles. By employment of virus replicon particles or infectious RNA which can replicate but not form de novo virions, we exclude a transfer of virus from the donor cell to the pDC. pDC activation by infected cells was mediated by a contact-dependent RNA transfer to pDC, which was sensitive to a TLR7 inhibitor. This was inhibited by drugs affecting the cytoskeleton and membrane cholesterol. We further demonstrate that a unique viral protein with ribonuclease activity, the viral Erns protein of pestiviruses, efficiently prevented this process. This required intact ribonuclease function in intracellular compartments. We propose that this pathway of activation could be of particular importance for viruses which tend to be mostly cell-associated, cause persistent infection, and are non-cytopathogenic.

Efficient sensing of infected cells in absence of virus particles by plasmacytoid dendritic is blocked by the viral ribonuclease Erns (P6114)

Abstract Plasmacytoid dendritic cells (pDC) have been shown to efficiently sense HCV- or HIV-infected cells. Here we demonstrate for classical swine fever virus, a member of the Flaviviridae, that this process is much more efficient in terms of interferon-alpha induction when compared to direct stimulation by virus particles. By employment of virus replicon particles which can replicate but not form de novo virions, we exclude a transfer of virus from the donor cell to the pDC. pDC activation by infected cells to pDC was mediated by a contact-dependent RNA transfer to TLR7, requiring intact cytoskeleton, lipid rafts, but not involving membrane vesicles such as exosomes. We further demonstrate that a unique viral protein with ribonuclease activity, the Erns protein of pestiviruses, efficiently prevented this process. This required intact ribonuclease function in intracellular compartments. The present study underlines the importance of pDC activation by infected cells and identifies a novel pathway of virus escaping the interferon system. This pathway of activation could be of particular importance for viruses which are cell-associated and cause persistent infection. Considering that Erns is required for pestiviruses to establish persistent infection of foetuses after transplacental virus transmission resulting in the development of immunotolerant animals, this report also points on a possible role of pDC in preventing immunotolerance after viral infection of foetuses.

Structural Transitions and Enzymatic Function of Ribonuclease A Encapsulated in Transparent Porous Silica Gel

Abstract Transparent porous silica gel encapsulating ribonuclease A (RNase A) in nanoscale pores was prepared on the wall of a quartz cell. The UV and CD spectral changes upon heating showed that the protein unfolds in the pores with apparent heat denaturation temperatures (Tm) at 60 to 64 °C, which are slightly lower than that in solution. The unfolding was irreversible due probably to strong interaction of the unfolded species with the surface of the pores upon prolonged treatment at high temperatures. However, when the gel was subjected to repeated temperature-jump experiments between 40 and 75 °C, RNase A reversibly unfolded and refolded in the pores. The unfolding was not apparently decelerated but the refolding was remarkably slowed. The protein refolded in the porous gel exhibited enzymatic activity toward cytidine 2′,3′-cyclic monophosphate (c-CMP), indicating that small molecules, such as c-CMP and its hydrolyzed species, can go through the holes of the silica gel. These results demonstrated not only that the wet porous gel is useful for the heat denaturation study of proteins but also that the gel encapsulating a protein can be applied as a soft material with enzymatic function.

The Tumor Marker Human Placental Protein 11 Is an Endoribonuclease

Human PP11 (placental protein 11) was previously described as a serine protease specifically expressed in the syncytiotrophoblast and in numerous tumor tissues. Several PP11-like proteins were annotated in distantly related organisms, such as worms and mammals, suggesting their involvement in evolutionarily conserved processes. Based on sequence similarity, human PP11 was included in a protein family whose characterized members are XendoU, a Xenopus laevis endoribonuclease involved in small nucleolar RNA processing, and Nsp15, an endoribonuclease essential for coronavirus replication. Here we show that the bacterially expressed human PP11 displays RNA binding capability and cleaves single stranded RNA in a Mn(2+)-dependent manner at uridylates, to produce molecules with 2',3'-cyclic phosphate ends. These features, together with structural and mutagenesis analyses, which identified the potential active site residues, reveal striking parallels to the amphibian XendoU and assign a ribonuclease function to PP11. This newly discovered enzymatic activity places PP11-like proteins in a completely new perspective.