The SC10-RNase promoter displays changes in DNA methylation patterns through pistil development in self-incompatible Nicotiana alata.

In Solanaceae, self-incompatibility is a genetic mechanism that prevents endogamy in plant populations. Expression of the S-determinants, S-RNase, and SLF, is tightly regulated during pistil and pollen development. However, the molecular mechanism of gene expression regulation in S-RNase-based self-incompatibility systems must be better understood. Here, we identified a 1.3 Kbp sequence upstream to the coding region of the functional SC10-RNase allele from the self-incompatible Nicotiana alata, which directs SC10-RNase expression in mature pistils. This SC10-RNase promoter includes a 300 bp region with minimal elements that sustain the SC10-RNase expression. Likewise, a fragment of a transposable element from the Gypsy family of retrotransposons is also present at the -320 bp position. Nevertheless, its presence does not affect the expression of the SC10-RNase in mature pistils. Additionally, we determined that the SC10-RNase promoter undergoes different DNA methylation states during pistil development, being the mCHH methylation context the most frequent close to the transcription start site at pistil maturity. We hypothesized that the Gypsy element at the SC10-RNase promoter might contribute to the DNA methylation remodeling on the three sequence contexts analyzed here. We propose that mCHH methylation enrichment and other regulatory elements in the S-RNase coding region regulate the specific and abundant SC10-RNase expression in mature pistils in N. alata.

Hormonal Signaling during dPCD: Cytokinin as the Determinant of RNase-Based Self-Incompatibility in Solanaceae.

Research into molecular mechanisms of self-incompatibility (SI) in plants can be observed in representatives of various families, including Solanaceae. Earlier studies of the mechanisms of S-RNase-based SI in petunia (Petunia hybrida E. Vilm.) demonstrate that programmed cell death (PCD) is an SI factor. These studies suggest that the phytohormon cytokinin (CK) is putative activator of caspase-like proteases (CLPs). In this work, data confirming this hypothesis were obtained in two model objects-petunia and tomato (six Solanaceae representatives). The exogenous zeatin treatment of tomato and petunia stigmas before a compatible pollination activates CLPs in the pollen tubes in vivo, as shown via the intravital imaging of CLP activities. CK at any concentration slows down the germination and growth of petunia and tomato male gametophytes both in vitro and in vivo; shifts the pH of the cytoplasm (PHc) to the acid region, thereby creating the optimal conditions for CLP to function and inhibiting the F-actin formation and/or destructing the cytoskeleton in pollen tubes to point foci during SI-induced PCD; and accumulates in style tissues during SI response. The activity of the ISOPENTENYLTRANSFERASE 5 (IPT5) gene at this moment exceeds its activity in a cross-compatible pollination, and the levels of expression of the CKX1 and CKX2 genes (CK OXIDASE/DEHYDROGENASE) are significantly lower in self-incompatible pollination. All this suggests that CK plays a decisive role in the mechanism underlying SI-induced PCD.

Ginkgo biloba Golden Leaf Extract (GGLE) Inhibits Melanoma Cell Invasion and Angiogenesis Through Inhibition of Angiogenin.

The popular dietary supplements of Ginkgo biloba (Ginkgo) products have been reported to have anti-cancer activities in multiple cellular and animal studies, with the benefits yet to be proven with clinical trials. The mechanisms of action are not clear, forming a barrier to investigation in Gingko-specific benefits to cancer patients, especially when combined with other therapies. Here we reported on the discovery of a novel mechanism by which a Ginkgo golden leaf extract (GGLE) inhibited melanoma cell invasion and angiogenesis. GGLE did not inhibit melanoma cells via direct cytotoxicity. Instead, GGLE significantly inhibited total RNase activities in melanoma cells under both normoxia and hypoxia conditions. The RNase angiogenin was induced twofolds by hypoxia, and the induction was significantly suppressed by GGLE treatment in a dose dependent manner. As a result of angiogenin inhibition, GGLE inhibited melanoma cell migration and invasion in a dose dependent manner. Conditioned media from melanoma cell culture sufficiently induced in vitro angiogenesis in human endothelial cells, whereas the conditioned media of GGLE-treated melanoma cells significantly inhibited this angiogenetic activity. This was accompanied with markedly reduced angiogenin concentrations in the GGLE-treated melanoma cell conditioned media. We concluded that, instead of direct cytotoxicity, GGLE inhibited angiogenin synthesis and secretion by melanoma cells, resulting in inhibition of tumor cell invasion and tumor-induced angiogenesis. This new mechanism opens the door for investigation in GGLE influencing tumor microenvironment, and warrants further investigation and validation in vivo.

BAG6-A from Fragaria viridis pollen modulates gametophyte development in diploid strawberry.

Male and female gametophyte development processes are essential steps in the life cycles of all land plants. Here, we characterized a gene, FviBAG6-A, screened from the Fragaria viridis (2 n = 2x=14) pollen cDNA library and physically interacted with S-RNase. Ubiquitinated of Sa-RNase might be determined by the interaction of FviBAG6-A in the ubiquitin-proteasome system during fertilization. We found that overexpression of FviBAG6-A in Arabidopsis caused shorter silique length, and decreased silique number. Moreover, overexpression of FviBAG6-A in Fragaria vesca (2 n = 2x=14) led to a greatly reduced seed number, with nearly 80% of the seeds aborted. Analyses of paraffin sections and reactive oxygen species (ROS) content revealed that the majority of severe pollen defects were likely due to the early degradation of the tapetum and middle layer as a result of ROS accumulation and abnormal development of the uninucleate megaspore mother. Moreover, the FviBAG6-A interact with the E3 ligase SIZ1 and contribute to the SUMOylation of FviBAG6-A , which may be induced by the high level of ROS content, further promoting gametophyte abortion in strawberry transgenic lines. This study characterized the FviBAG6-A and reveals its novel function in gametophyte development.

Detection of Ilyonectria pseudodestructans from Potato by RNase H-Dependent PCR (rhPCR) and rh-Quantitative PCR (rhqPCR).

Ilyonectria pseudodestructans, a plant pathogen that is known to cause root rot on fruit trees such as grapevine and apple, has recently been reported to also cause tuber decay in potato. The increasing risk of this pathogen on various horticultural crops makes it essential to develop a rapid and accurate detection method. In this study, an RNase H-dependent PCR (rhPCR) protocol and a modified probe-based rh-quantitative PCR (rhqPCR) protocol for I. pseudodestructans detection were developed. Both the forward and reverse primers for rhPCR and rhqPCR carry an RNA nucleotide at the site where a single-nucleotide polymorphism between I. pseudodestructans and strains of other Ilyonectria spp. is located, and the rhqPCR also contains a fluorescent-labeled target-specific probe. The primers were designed based on the sequence of the histone H3 gene and could amplify a DNA fragment of 73 bp. In the specificity test, by alignment via the BLASTn tool, the RNA nucleotide bases on both the forward and the reverse primers were identical to the corresponding genomic site of 16 of 17 (94.1%) database-available I. pseudodestructans strains, and different from 43 of 44 (97.7%) database-available strains of other Ilyonectria spp. When the rhPCR and rhqPCR protocols were applied on 11 I. pseudodestructans strains and 46 other strains of different species of plant pathogens, all of the I. pseudodestructans strains generated positive reactions whereas all of the other strains were negative, which indicated an excellent specificity of the primers. In the sensitivity test, the lowest DNA template amount for a positive reaction using the rhPCR and rhqPCR methods was 2 pg for I. pseudodestructans genomic DNA. When testing the rhqPCR method on gBlock, the lowest number of molecules for a positive reaction was six. These results indicated a high sensitivity of the protocol for I. pseudodestructans detection. To our knowledge, this is the first report of a probe-based rhqPCR to be applied to plant disease diagnosis; in addition, this is also the first rapid molecular protocol to detect I. pseudodestructans. The new rhPCR and rhqPCR methods have a potential to be applied by plant disease diagnostic labs for their routine work.

Self S-RNase inhibits ABF-LRX signaling to arrest pollen tube growth to achieve self-incompatibility in pear.

Gametophytic self-incompatibility (GSI) has been widely studied in flowering plants, but studies of the mechanisms underlying pollen tube growth arrest by self S-RNase in GSI species are limited. In the present study, two leucine-rich repeat extensin genes in pear (Pyrus bretschneideri), PbLRXA2.1 and PbLRXA2.2, were identified based on transcriptome and quantitative real-time PCR analyses. The expression levels of these two LRX genes were significantly higher in the pollen grains and pollen tubes of the self-compatible cultivar 'Jinzhui' (harboring a spontaneous bud mutation) than in those of the self-incompatible cultivar 'Yali'. Both PbLRXA2.1 and PbLRXA2.2 stimulated pollen tube growth and attenuated the inhibitory effects of self S-RNase on pollen tube growth by stabilizing the actin cytoskeleton and enhancing cell wall integrity. These results indicate that abnormal expression of PbLRXA2.1 and PbLRXA2.2 is involved in the loss of self-incompatibility in 'Jinzhui'. The PbLRXA2.1 and PbLRXA2.2 promoters were directly bound by the ABRE-binding factor PbABF.D.2. Knockdown of PbABF.D.2 decreased PbLRXA2.1 and PbLRXA2.2 expression and inhibited pollen tube growth. Notably, the expression of PbLRXA2.1, PbLRXA2.2, and PbABF.D.2 was repressed by self S-RNase, suggesting that self S-RNase can arrest pollen tube growth by restricting the PbABF.D.2-PbLRXA2.1/PbLRXA2.2 signal cascade. These results provide novel insight into pollen tube growth arrest by self S-RNase.

Boron Clusters as Enhancers of RNase H Activity in the Smart Strategy of Gene Silencing by Antisense Oligonucleotides.

Boron cluster-conjugated antisense oligonucleotides (B-ASOs) have already been developed as therapeutic agents with "two faces", namely as potential antisense inhibitors of gene expression and as boron carriers for boron neutron capture therapy (BNCT). The previously observed high antisense activity of some B-ASOs targeting the epidermal growth factor receptor (EGFR) could not be rationally assigned to the positioning of the boron cluster unit: 1,2-dicarba-closo-dodecaborane (0), [(3,3'-Iron-1,2,1',2'-dicarbollide) (1-), FESAN], and dodecaborate (2-) in the ASO chain and its structure or charge. For further understanding of this observation, we performed systematic studies on the efficiency of RNase H against a series of B-ASOs models. The results of kinetic analysis showed that pyrimidine-enriched B-ASO oligomers activated RNase H more efficiently than non-modified ASO. The presence of a single FESAN unit at a specific position of the B-ASO increased the kinetics of enzymatic hydrolysis of complementary RNA more than 30-fold compared with unmodified duplex ASO/RNA. Moreover, the rate of RNA hydrolysis enhanced with the increase in the negative charge of the boron cluster in the B-ASO chain. In conclusion, a "smart" strategy using ASOs conjugated with boron clusters is a milestone for the development of more efficient antisense therapeutic nucleic acids as inhibitors of gene expression.

Genome-Wide Analysis of the RNase T2 Family and Identification of Interacting Proteins of Four ClS-RNase Genes in 'XiangShui' Lemon.

S-RNase plays vital roles in the process of self-incompatibility (SI) in Rutaceae plants. Data have shown that the rejection phenomenon during self-pollination is due to the degradation of pollen tube RNA by S-RNase. The cytoskeleton microfilaments of pollen tubes are destroyed, and other components cannot extend downwards from the stigma and, ultimately, cannot reach the ovary to complete fertilisation. In this study, four S-RNase gene sequences were identified from the 'XiangShui' lemon genome and ubiquitome. Sequence analysis revealed that the conserved RNase T2 domains within S-RNases in 'XiangShui' lemon are the same as those within other species. Expression pattern analysis revealed that S3-RNase and S4-RNase are specifically expressed in the pistils, and spatiotemporal expression analysis showed that the S3-RNase expression levels in the stigmas, styles and ovaries were significantly higher after self-pollination than after cross-pollination. Subcellular localisation analysis showed that the S1-RNase, S2-RNase, S3-RNase and S4-RNase were found to be expressed in the nucleus according to laser confocal microscopy. In addition, yeast two-hybrid (Y2H) assays showed that S3-RNase interacted with F-box, Bifunctional fucokinase/fucose pyrophosphorylase (FKGP), aspartic proteinase A1, RRP46, pectinesterase/pectinesterase inhibitor 51 (PME51), phospholipid:diacylglycerol acyltransferase 1 (PDAT1), gibberellin receptor GID1B, GDT1-like protein 4, putative invertase inhibitor, tRNA ligase, PAP15, PAE8, TIM14-2, PGIP1 and p24beta2. Moreover, S3-RNase interacted with TOPP4. Therefore, S3-RNase may play an important role in the SI of 'XiangShui' lemon.

Polyamines Involved in Regulating Self-Incompatibility in Apple.

Apple exhibits typical gametophytic self-incompatibility, in which self-S-RNase can arrest pollen tube growth, leading to failure of fertilization. To date, there have been few studies on how to resist the toxicity of self-S-RNase. In this study, pollen tube polyamines were found to respond to self-S-RNase and help pollen tubes defend against self-S-RNase. In particular, the contents of putrescine, spermidine, and spermine in the pollen tube treated with self-S-RNase were substantially lower than those treated with non-self-S-RNase. Further analysis of gene expression of key enzymes in the synthesis and degradation pathways of polyamines found that the expression of DIAMINE OXIDASE 4 (MdDAO4) as well as several polyamine oxidases such as POLYAMINE OXIDASES 3 (MdPAO3), POLYAMINE OXIDASES 4 (MdPAO4), and POLYAMINE OXIDASES 6 (MdPAO6) were significantly up-regulated under self-S-RNase treatment, resulting in the reduction of polyamines. Silencing MdPAO6 in pollen tubes alleviates the inhibitory effect of self-S-RNase on pollen tube growth. In addition, exogenous polyamines also enhance pollen tube resistance to self-S-RNase. Transcriptome sequencing data found that polyamines may communicate with S-RNase through the calcium signal pathway, thereby regulating the growth of the pollen tubes. To summarize, our results suggested that polyamines responded to the self-incompatibility reaction and could enhance pollen tube tolerance to S-RNase, thus providing a potential way to break self-incompatibility in apple.

Deficiency of ER Ca2+ sensor STIM1 in AgRP neurons confers protection against dietary obesity.

Store-operated calcium entry (SOCE) is pivotal in maintaining intracellular Ca2+ level and cell function; however, its role in obesity development remains largely unknown. Here, we show that the stromal interaction molecule 1 (Stim1), an endoplasmic reticulum (ER) Ca2+ sensor for SOCE, is critically involved in obesity development. Pharmacological blockade of SOCE in the brain, or disruption of Stim1 in hypothalamic agouti-related peptide (AgRP)-producing neurons (ASKO), significantly ameliorates dietary obesity and its associated metabolic disorders. Conversely, constitutive activation of Stim1 in AgRP neurons leads to an obesity-like phenotype. We show that the blockade of SOCE suppresses general translation in neuronal cells via the 2',5'-oligoadenylate synthetase 3 (Oas3)-RNase L signaling. While Oas3 overexpression in AgRP neurons protects mice against dietary obesity, deactivation of RNase L in these neurons significantly abolishes the effect of ASKO. These findings highlight an important role of Stim1 and SOCE in the development of obesity.

Primary restriction of S-RNase cytotoxicity by a stepwise ubiquitination and degradation pathway in Petunia hybrida.

In self-incompatible Petunia species, the pistil S-RNase acts as cytotoxin to inhibit self-pollination but is polyubiquitinated by the pollen-specific nonself S-locus F-box (SLF) proteins and subsequently degraded by the ubiquitin-proteasome system (UPS), allowing cross-pollination. However, it remains unclear how S-RNase is restricted by the UPS. Using biochemical analyses, we first show that Petunia hybrida S3 -RNase is largely ubiquitinated by K48-linked polyubiquitin chains at three regions, R I, R II and R III. R I is ubiquitinated in unpollinated, self-pollinated and cross-pollinated pistils, indicating its occurrence before PhS3 -RNase uptake into pollen tubes, whereas R II and R III are exclusively ubiquitinated in cross-pollinated pistils. Transgenic analyses showed that removal of R II ubiquitination resulted in significantly reduced seed sets from cross-pollination and that of R I and R III to a lesser extent, indicating their increased cytotoxicity. Consistent with this, the mutated R II of PhS3 -RNase resulted in a marked reduction of its degradation, whereas that of R I and R III resulted in less reduction. Taken together, we demonstrate that PhS3 -RNase R II functions as a major ubiquitination region for its destruction and R I and R III as minor ones, revealing that its cytotoxicity is primarily restricted by a stepwise UPS mechanism for cross-pollination in P. hybrida.

In Vitro Identification and In Vivo Confirmation of Inhibitors for Sweet Potato Chlorotic Stunt Virus RNA Silencing Suppressor, a Viral RNase III.

Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield losses all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as an RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases.IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.

Flavonoids and Acid-Hydrolysis derivatives of Neo-Clerodane diterpenes from Teucrium flavum subsp. glaucum as inhibitors of the HIV-1 reverse transcriptase-associated RNase H function.

Bioassay-guided fractionation of the ethyl acetate extract from Teucrium flavum subsp. glaucum, endowed with inhibitory activity towards the HIV-1 reverse transcriptase-associated RNase H function, led to the isolation of salvigenin (1), cirsimaritin (2) and cirsiliol (3) along with the neo-clerodanes teuflavin (4) and teuflavoside (5). Acid hydrolysis of the inactive teuflavoside provided three undescribed neo-clerodanes, flavuglaucins A-C (7-9) and one known neo-clerodane (10). Among all neo-clerodanes, flavuglaucin B showed the highest inhibitory activity towards RNase H function with a IC50 value of 9.1 µM. Molecular modelling and site-directed mutagenesis analysis suggested that flavuglaucin B binds into an allosteric pocket close to RNase H catalytic site. This is the first report of clerodane diterpenoids endowed with anti-reverse transcriptase activity. Neo-clerodanes represent a valid scaffold for the development of a new class of HIV-1 RNase H inhibitors.

Targeting HIV-1 RNase H: N'-(2-Hydroxy-benzylidene)-3,4,5-Trihydroxybenzoylhydrazone as Selective Inhibitor Active against NNRTIs-Resistant Variants.

HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations. Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants. The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor. Here, we characterize the mode of action of N'-(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound 13), an N-acylhydrazone derivative that inhibited viral replication (EC50 = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 13 targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being >20-fold less potent against the RT polymerase activity. Docking calculations revealed that compound 13 binds within the RNase H domain in a position different from other selective RNase H inhibitors; site-directed mutagenesis studies revealed interactions with conserved amino acid within the RNase H domain, suggesting that compound 13 can be taken as starting point to generate a new series of more potent RNase H selective inhibitors active against circulating drug-resistant variants.

NaTrxh is an essential protein for pollen rejection in Nicotiana by increasing S-RNase activity.

In self-incompatible Solanaceae, the pistil protein S-RNase contributes to S-specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S-RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S-RNase in vitro. Here, we show that expressing a redox-inactive mutant, NaTrxhSS , suppresses both S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys155 -Cys185 disulphide bond of SC10 -Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S-RNase activity by NaTrxh helps to explain why S-RNase alone could be insufficient for pollen rejection.

Polydopamine-coated nucleic acid nanogel for siRNA-mediated low-temperature photothermal therapy.

Photothermal therapy (PTT) normally requires to maintain the temperature of tumor lesions above 50 °C, which potentially induces local inflammation and tumor metastasis. To avoid these side effects, it is vital to achieve effective antitumor efficacy at relatively low temperature (42-45 °C) during the PTT treatment. Herein, we design a polydopamine (PDA)-coated nucleic acid nanogel as a therapeutic complex for siRNA-mediated low-temperature PTT. First, siRNAs that target the heat-shock-protein 70 (Hsp70) serve as crosslinkers to guide the DNA-grafted polycaprolactone (DNA-g-PCL) assemble into nanosized hydrogel particles through nucleic acid hybridization. Thereafter, the obtained siRNA-embedded nanogels are further coated with a thin layer of polydopamine, which not only protects the nanogels against enzymatic degradation but also endows the nanogels with excellent photothermal conversion capacity under near infrared (NIR) light irradiation. After surface PEGylation, this triple shield siRNA delivery complex shows the capability of effective ablating the tumor under relatively mild condition.

RNase Protection Assay.

Unbound, single-stranded RNA can be digested by RNase (A or T1) to ribonucleotides, whereas double-stranded RNA is not digested by RNase. Based on this principle, the RNase Protection Assay (RPA) is used to validate chimeric RNAs. Importantly, this assay does not employ reverse transcription (RT), thus avoiding potential false-positive results which could occur during RT such as template-switching. We first generate RNA probes with 32phosphate (P) or biotin that are complementary to the predicted nucleotide sequence of the chimeric RNA, then hybridize them to RNA samples. The labeled RNA probes can bind specifically with the target chimeric RNA in order to form double-stranded RNA. This newly formed RNA is resistant to digestion by RNase and therefore can be identified by high-resolution, denaturing polyacrylamide gel electrophoresis.

A Pathogenesis-Related Protein-Like Gene Is Involved in the Panax notoginseng Defense Response to the Root Rot Pathogen.

Pathogenesis-related proteins (PRs) are a class of proteins that accumulate in response to biotic and abiotic stresses to protect plants from damage. In this study, a gene encoding a PR-like protein (PnPR-like) was isolated from Panax notoginseng, which is used in traditional Chinese herbal medicines. An analysis of gene expression in P. notoginseng indicated that PnPR-like was responsive to an infection by the root rot pathogen Fusarium solani. The expression of this gene was induced by several signaling molecules, including methyl jasmonate, ethephon, hydrogen peroxide, and salicylic acid. The PnPR-like-GFP fusion gene was transiently expressed in onion (Allium cepa) epidermal cells, which revealed that PnPR-like is a cytoplasmic protein. The purified recombinant PnPR-like protein expressed in Escherichia coli had antifungal effects on F. solani and Colletotrichum gloeosporioides as well as inhibited the spore germination of F. solani. Additionally, the in vitro ribonuclease (RNase) activity of the recombinant PnPR-like protein was revealed. The PnPR-like gene was inserted into tobacco (Nicotiana tabacum) to verify its function. The gene was stably expressed in T2 transgenic tobacco plants, which exhibited more RNase activity and greater disease resistance than the wild-type tobacco. Moreover, the transient expression of hairpin RNA targeting PnPR-like in P. notoginseng leaves increased the susceptibility to F. solani and decreased the PnPR-like expression level. In conclusion, the cytoplasmic protein PnPR-like, which has RNase activity, is involved in the P. notoginseng defense response to F. solani.

Actinomycin D-Activated RNase L Promotes H2A.X/H2B-Mediated DNA Damage and Apoptosis in Lung Cancer Cells.

Background: Chemotherapy is an essential component for comprehensive cancer treatment, while drug resistance usually fails therapy. DNA repair mechanism of cancer cells restrains the efficacy of therapeutics targeting DNA damage. Investigating target-inducing irreversible cell death of cancer cells may be promising. Methods: The present study used lung cancer cell lines, transplanted tumor model of lung cancers derived from patients with lung adenocarcinoma, and molecular experiments to investigate the effects and mechanism of Actinomycin D (Act D)-activated RNase L in lung canceers. Results: We report that RNase L, when activated by Act D, induces Caspase-3/PARP activation. The latter further enables ROCK-1 to initiate subsequent membrane blebbing and, meanwhile, result in DNA cleavage and cell cycle arrest mediated by H2A.X/H2B-p21 axis, leading to irreversible DNA damage, and apoptosis of lung cancer cells. The present study highlighted the crucial role of RNase L in triggering apoptosis mechanism through the Caspase-3/ROCK-1/PARP/H2A.X+H2B/p21 axis during Act D treatment. Moreover, activation of RNase L suppressed the tumor formation and the induction of lung cancer stem cells. Conclusion: This study unveiled the regulatory function and related mechanism of RNase L and implied the promising application of therapeutics targeting RNase L in lung cancer.

Synthesis, biophysical properties, and RNase H activity of 6'-difluoro[4.3.0]bicyclo-DNA.

Here we present the synthesis, the biophysical properties, and the RNase H profile of 6'-difluorinated [4.3.0]bicyclo-DNA (6'-diF-bc4,3-DNA). The difluorinated thymidine phosphoramidite building block was synthesized starting from an already known gem-difluorinated tricyclic glycal. This tricyclic siloxydifluorocyclopropane was converted into the [4.3.0]bicyclic nucleoside via cyclopropane ring-opening through the addition of an electrophilic iodine during the nucleosidation step followed by reduction. The gem-difluorinated bicyclic nucleoside was then converted into the corresponding phosphoramidite building block which was incorporated into oligonucleotides. Thermal denaturation experiments of these oligonucleotides hybridized to complementary DNA or RNA disclosed a significant destabilization of both duplex types (ΔT m/mod = -1.6 to -5.5 °C). However, in the DNA/RNA hybrid the amount of destabilization could be reduced by multiple insertions of the modified unit. In addition, CD spectroscopy of the oligonucleotides hybridized to RNA showed a similar structure than the natural DNA/RNA duplex. Furthermore, since the structural investigation on the nucleoside level by X-ray crystallography and ab initio calculations pointed to a furanose conformation in the southern region, a RNase H cleavage assay was conducted. This experiment revealed that the oligonucleotide containing five modified units was able to elicit the RNase H-mediated cleavage of the complementary RNA strand.