RESUMEN
BACKGROUND: Human immunodeficiency virus (HIV)-1 infection can activate the expression of human endogenous retroviruses (HERVs), particularly HERV-K (HML-2). HIV controllers (HICs) are rare people living with HIV (PLWHs) who naturally control HIV-1 replication and overexpress some cellular restriction factors that negatively regulate the LTR-driven transcription of HIV-1 proviruses. OBJECTIVES: To understand the ability of HICs to control the expression of endogenous retroviruses. METHODS: We measured endogenous retrovirus type K6 (ERVK-6) RNA expression in peripheral blood mononuclear cells (PBMCs) of HICs (n = 23), antiretroviral (ART)-suppressed subjects (n = 8), and HIV-1-negative (NEG) individuals (n = 10) and correlated the transcript expression of ERVK-6 with multiple HIV-1 cellular restriction factors. FINDINGS: Our study revealed that ERVK-6 RNA expression in PBMCs from HICs was significantly downregulated compared with that in both the ART and NEG control groups. Moreover, we detected that ERVK-6 RNA levels in PBMCs across all groups were negatively correlated with the expression levels of p21 and MCPIP1, two cellular restriction factors that limit the activation of macrophages and T cells by downregulating the activity of NF-kB. MAIN CONCLUSIONS: These findings support the hypothesis that HICs activate innate antiviral mechanisms that may simultaneously downregulate the transcription of both exogenous (HIV-1) and endogenous (ERVK-6) retroviruses. Future studies with larger cohorts should be performed to confirm this hypothesis and to explore the role of p21 and MCPIP1 in regulating HERV-K expression in physiological and pathological conditions.
Asunto(s)
Retrovirus Endógenos , Infecciones por VIH , VIH-1 , ARN Viral , Ribonucleasas , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios de Casos y Controles , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Retrovirus Endógenos/genética , Retrovirus Endógenos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Infecciones por VIH/genética , VIH-1/genética , VIH-1/inmunología , Inmunidad Innata/genética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ribonucleasas/genética , Ribonucleasas/metabolismo , ARN Viral/genética , Factores de Transcripción/genética , Replicación Viral/genéticaRESUMEN
The leafhopper Dalbulus maidis is a harmful pest that causes severe damage to corn crops. Conventional chemical pesticides have negative environmental impacts, emphasizing the need for alternative solutions. RNA interference (RNAi) is a more specific and environmentally friendly method for controlling pests and reducing the negative impacts of current pest management practices. Previous studies have shown that orally administered double-stranded RNA (dsRNA) is less effective than injection protocols in silencing genes. This study focuses on identifying and understanding the role of double-stranded ribonucleases (dsRNases) in limiting the efficiency of oral RNAi in D. maidis. Three dsRNases were identified and characterized, with Dmai-dsRNase-2 being highly expressed in the midgut and salivary glands. An ex vivo degradation assay revealed significant nuclease activity, resulting in high instability of dsRNA when exposed to tissue homogenates. Silencing Dmai-dsRNase-2 improved the insects' response to the dsRNA targeting the gene of interest, providing evidence of dsRNases involvement in oral RNAi efficiency. Therefore, administering both dsRNase-specific and target gene-specific-dsRNAs simultaneously is a promising approach to increase the efficiency of oral RNAi and should be considered in future control strategies.
Asunto(s)
Hemípteros , Ribonucleasas , Animales , Ribonucleasas/genética , Ribonucleasas/metabolismo , Interferencia de ARN , Zea mays/genética , Zea mays/metabolismo , Hemípteros/genética , Hemípteros/metabolismo , Insectos/genética , ARN Bicatenario/genéticaRESUMEN
Type 1 conventional dendritic cells (cDC1s) are leukocytes competent to coordinate antiviral immunity, and thus, the intracellular mechanisms controlling cDC1 function are a matter of intense research. The unfolded protein response (UPR) sensor IRE1 and its associated transcription factor XBP1s control relevant functional aspects in cDC1s including antigen cross-presentation and survival. However, most studies connecting IRE1 and cDC1 function are undertaken in vivo. Thus, the aim of this work is to elucidate whether IRE1 RNase activity can also be modeled in cDC1s differentiated in vitro and reveal the functional consequences of such activation in cells stimulated with viral components. Our data show that cultures of optimally differentiated cDC1s recapitulate several features of IRE1 activation noticed in in vivo counterparts and identify the viral analog Poly(I:C) as a potent UPR inducer in the lineage. In vitro differentiated cDC1s display constitutive IRE1 RNase activity and hyperactivate IRE1 RNase upon genetic deletion of XBP1s, which regulates production of the proinflammatory cytokines IL-12p40, TNF-α and IL-6, Ifna and Ifnb upon Poly(I:C) stimulation. Our results show that a strict regulation of the IRE1/XBP1s axis regulates cDC1 activation to viral agonists, expanding the scope of this UPR branch in potential DC-based therapies.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Respuesta de Proteína Desplegada , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/metabolismo , Ribonucleasas/metabolismoRESUMEN
In cancer, activation of the IRE1/XBP1s axis of the unfolded protein response (UPR) promotes immunosuppression and tumor growth, by acting in cancer cells and tumor infiltrating immune cells. However, the role of IRE1/XBP1s in dendritic cells (DCs) in tumors, particularly in conventional type 1 DCs (cDC1s) which are cellular targets in immunotherapy, has not been fully elucidated. Here, we studied the role of IRE1/XBP1s in subcutaneous B16/B78 melanoma and MC38 tumors by generating loss-of-function models of IRE1 and/or XBP1s in DCs or in cDC1s. Data show that concomitant deletion of the RNase domain of IRE1 and XBP1s in DCs and cDC1s does not influence the kinetics of B16/B78 and MC38 tumor growth or the effector profile of tumor infiltrating T cells. A modest effect is observed in mice bearing single deletion of XBP1s in DCs, which showed slight acceleration of melanoma tumor growth and dysfunctional T cell responses, however, this effect was not recapitulated in animals lacking XBP1 only in cDC1s. Thus, evidence presented here argues against a general pro-tumorigenic role of the IRE1/XBP1s pathway in tumor associated DC subsets.
Asunto(s)
Melanoma Experimental , Ribonucleasas , Ratones , Animales , Ribonucleasas/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Inmunidad Adaptativa , Ribonucleasa Pancreática/metabolismo , Melanoma Experimental/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Células DendríticasRESUMEN
The PumAB type-II toxin-antitoxin (TA) system is encoded by pumAB genes that are organized into an operon. This system is encoded by the pUM505 plasmid, isolated from a Pseudomonas aeruginosa clinical strain. The pumA gene encodes a putative RelE toxin protein (toxic component), whereas the pumB gene encodes a putative HTH antitoxin protein. The expression of the PumAB system in Escherichia coli confers plasmid stability. In addition, PumA toxin overexpression in P. aeruginosa possesses the capability to increase bacterial virulence, an effect that is neutralized by the PumB antitoxin. The aim of this study was to establish the mechanism of regulation of the PumAB toxin-antitoxin system from pUM505. By an in silico analysis of the putative regulatory elements, we identified two putative internal promoters, PpumB and PpumB-AlgU (in addition to the already reported PpumAB), located upstream of pumB. By RT-qPCR assays, we determined that the pumAB genes are transcribed differentially, in that the mRNA of pumB is more abundant than the pumA transcript. We also observed that pumB could be expressed individually and that its mRNA levels decreased under oxidative stress, during individual expression as well as co-expression of pumAB. However, under stressful conditions, the pumA mRNA levels were not affected. This suggests the negative regulation of pumB by stressful conditions. The PumB purified protein was found to bind to a DNA region located between the PpumAB and the pumA coding region, and PumA participates in PumB binding, suggesting that a PumA-PumB complex co-regulates the transcription of the pumAB operon. Interestingly, the pumA mRNA levels decreased after incubation in vitro with PumB protein. This effect was repressed by ribonuclease inhibitors, suggesting that PumB could function as an RNAse toward the mRNA of the toxin. Taken together, we conclude that the PumAB TA system possesses multiple mechanisms to regulate its expression, as well as that the PumB antitoxin generates a decrease in the mRNA toxin levels, suggesting an RNase function. Our analysis provides new insights into the understanding of the control of TA systems from mobile plasmid-encoded genes from a human pathogen.
Asunto(s)
Antitoxinas , Toxinas Bacterianas , Sistemas Toxina-Antitoxina , Humanos , Antitoxinas/genética , Antitoxinas/metabolismo , Toxinas Bacterianas/genética , Sistemas Toxina-Antitoxina/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , ARN Mensajero , Ribonucleasas/genética , Ribonucleasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Genoma Viral , ARN Viral , Inactivación de Virus , Animales , Células Cultivadas , Chlorocebus aethiops , Efecto Citopatogénico Viral , ADN Complementario/biosíntesis , Virus de la Encefalitis Equina del Este/genética , Virus de la Encefalitis Equina del Este/fisiología , Virus de la Encefalitis Equina Venezolana/fisiología , Virus de la Encefalitis Equina del Oeste/genética , Virus de la Encefalitis Equina del Oeste/fisiología , ARN Viral/química , ARN Viral/fisiología , Ribonucleasas/metabolismo , Células VeroRESUMEN
Thioredoxins are regulatory proteins that reduce disulfide bonds on target proteins. NaTrxh, which belongs to the plant thioredoxin family h subgroup 2, interacts and reduces the S-RNase enhancing its ribonuclease activity seven-fold, resulting an essential protein for pollen rejection inNicotiana.Here, the crystal structure of NaTrxh at 1.7 Å by X-ray diffraction is reported. NaTrxh conserves the typical fold observed in other thioredoxins from prokaryotes and eukaryotes, but it contains extensions towards both N- and C-termini.The NaTrxh N-terminal extension participates in the reduction of S-RNase, and in the structure reported here, this is orientated towards the reactive site. The interaction between SF11-RNase and the NaTrxh N-terminal was simulated and the short-lived complex observed lasted for a tenth of ns. Moreover, we identified certain amino acids as SF11-RNase-E155 and NaTrxh-M104 as good candidates to contribute to the stability of the complex. Furthermore, we simulated the reduction of the C153-C186 SF11-RNase disulfide bond and observed subtle changes that affect the entire core, which might explain the increase in the ribonuclease activity of S-RNase when it is reduced by NaTrxh.
Asunto(s)
Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Ribonucleasas/metabolismo , Sitios de Unión/fisiología , Eucariontes/metabolismo , Células Procariotas/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
BACKGROUND: Some multifunctional cellular proteins, as the monocyte chemotactic protein-induced protein 1 (ZC3H12A/MCPIP1) and the cyclin-dependent kinase inhibitor CDKN1A/p21, are able to modulate the cellular susceptibility to the human immunodeficiency virus type 1 (HIV-1). Several studies showed that CDKN1A/p21 is expressed at high levels ex vivo in cells from individuals who naturally control HIV-1 replication (HIC) and a recent study supports a coordinate regulation of ZC3H12A/MCPIP1 and CDKN1A/p21 transcripts in a model of renal carcinoma cells. Here, we explored the potential associations between mRNA expression of ZC3H12A/MCPIP1 and CDKN1A/p21 in HIC sustaining undetectable (elite controllers-EC) or low (viremic controllers-VC) viral loads. RESULTS: We found a selective upregulation of ZC3H12A/MCPIP1 and CDKN1A/p21 mRNA levels in PBMC from HIC compared with both ART-suppressed and HIV-negative control groups (P≤ 0.02) and higher MCPIP1 and p21 proteins levels in HIC than in HIV-1 negative subjects. There was a moderate positive correlation (r ≥ 0.57; P ≤ 0.014) between expressions of both transcripts in HIC and in HIC combined with control groups. We found positive correlations between the mRNA level of CDKN1A/p21 with activated CD4+ T cells levels in HIC (r ≥ 0.53; P ≤ 0.017) and between the mRNA levels of both CDKN1A/p21 (r = 0.74; P = 0.005) and ZC3H12A/MCPIP1 (r = 0.58; P = 0.040) with plasmatic levels of sCD14 in EC. Reanalysis of published transcriptomic data confirmed the positive association between ZC3H12A/MCPIP1 and CDKN1A/p21 mRNA levels in CD4+ T cells and monocytes from disparate cohorts of HIC and other HIV-positive control groups. CONCLUSIONS: These data show for the first time the simultaneous upregulation of ZC3H12A/MCPIP1 and CDKN1A/p21 transcripts in the setting of natural suppression of HIV-1 replication in vivo and the positive correlation of the expression of these cellular factors in disparate cohorts of HIV-positive individuals. The existence of a common regulatory pathway connecting ZC3H12A/MCPIP1 and CDKN1A/p21 could have a synergistic effect on HIV-1 replication control and pharmacological manipulation of these multifunctional host factors may open novel therapeutic perspectives to prevent HIV-1 replication and disease progression.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Infecciones por VIH/inmunología , VIH-1/fisiología , Ribonucleasas/metabolismo , Factores de Transcripción/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Leucocitos Mononucleares/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , ARN Mensajero/metabolismo , Ribonucleasas/genética , Factores de Transcripción/genética , Regulación hacia Arriba , Carga ViralRESUMEN
Fibrillarin is a highly conserved nucleolar methyltransferase responsible for ribosomal RNA methylation across evolution from Archaea to humans. It has been reported that fibrillarin is involved in the methylation of histone H2A in nucleoli and other processes, including viral progression, cellular stress, nuclear shape, and cell cycle progression. We show that fibrillarin has an additional activity as a ribonuclease. The activity is affected by phosphoinositides and phosphatidic acid and insensitive to ribonuclease inhibitors. Furthermore, the presence of phosphatidic acid releases the fibrillarin-U3 snoRNA complex. We show that the ribonuclease activity localizes to the GAR (glycine/arginine-rich) domain conserved in a small group of RNA interacting proteins. The introduction of the GAR domain occurred in evolution in the transition from archaea to eukaryotic cells. The interaction of this domain with phospholipids may allow a phase separation of this protein in nucleoli.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Fosfolípidos/metabolismo , Ribonucleasas/química , Ribonucleasas/metabolismo , Proteínas Cromosómicas no Histona/genética , Células HeLa , Humanos , Mutación/genética , Dominios Proteicos , ARN Nucleolar Pequeño/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleasas/genética , Ribonucleoproteínas/metabolismo , Relación Estructura-ActividadRESUMEN
KEY MESSAGE: The early flowering system HSP::AtFT allowed a fast evaluation of a gene containment system based on the construct PsEND1::barnase-barstar for poplar. Transgenic lines showed disturbed pollen development and sterility. Vertical gene transfer through pollen flow from transgenic or non-native plant species into their crossable natural relatives is a major concern. Gene containment approaches have been proposed to reduce or even avoid gene flow among tree species. However, evaluation of genetic containment strategies for trees is very difficult due to the long-generation times. Early flowering induction would allow faster evaluation of genetic containment in this case. Although no reliable methods were available for the induction of fertile flowers in poplar, recently, a new early flowering approach was developed. In this study, early flowering poplar lines containing the gene construct PsEND1::barnase-barstar were obtained. The PsEND1 promoter was chosen due to its early expression pattern, its versality and efficiency for generation of male-sterile plants fused to the barnase gene. RT-PCRs confirmed barnase gene activity in flowers, and pollen development was disturbed, leading to sterile flowers. The system developed in this study represents a valuable tool for gene containment studies in forest tree species.
Asunto(s)
Proteínas Bacterianas/genética , Flores/crecimiento & desarrollo , Edición Génica/métodos , Infertilidad Vegetal/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Polen/crecimiento & desarrollo , Populus/crecimiento & desarrollo , Ribonucleasas/genética , Proteínas de Arabidopsis/genética , Proteínas Bacterianas/metabolismo , Flores/genética , Flores/metabolismo , Flores/efectos de la radiación , Regulación de la Expresión Génica de las Plantas , Flujo Génico , Vectores Genéticos , Respuesta al Choque Térmico , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Polen/genética , Populus/genética , Populus/metabolismo , Populus/efectos de la radiación , Regiones Promotoras Genéticas , Ribonucleasas/metabolismo , Temperatura , Transformación GenéticaRESUMEN
Poly(A) tail shortening is a critical step in messenger RNA (mRNA) decay and control of gene expression. The carbon catabolite repressor 4 (CCR4)-associated factor 1 (CAF1) component of the CCR4-NOT deadenylase complex plays an essential role in mRNA deadenylation in most eukaryotes. However, while CAF1 has been extensively investigated in yeast and animals, its role in plants remains largely unknown. Here, we show that the Citrus sinensis CAF1 (CsCAF1) is a magnesium-dependent deadenylase implicated in resistance against the citrus canker bacteria Xanthomonas citri. CsCAF1 interacted with proteins of the CCR4-NOT complex, including CsVIP2, a NOT2 homologue, translin-associated factor X (CsTRAX) and the poly(A)-binding proteins CsPABPN and CsPABPC. CsCAF1 also interacted with PthA4, the main X. citri effector required for citrus canker elicitation. We also present evidence suggesting that PthA4 inhibits CsCAF1 deadenylase activity in vitro and stabilizes the mRNA encoded by the citrus canker susceptibility gene CsLOB1, which is transcriptionally activated by PthA4 during canker formation. Moreover, we show that an inhibitor of CsCAF1 deadenylase activity significantly enhanced canker development, despite causing a reduction in PthA4-dependent CsLOB1 transcription. These results thus link CsCAF1 with canker development and PthA4-dependent transcription in citrus plants.
Asunto(s)
Citrus sinensis/enzimología , Citrus sinensis/microbiología , Resistencia a la Enfermedad/inmunología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Ribonucleasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Citrus sinensis/genética , Citrus sinensis/inmunología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Magnesio/farmacología , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Poli A/metabolismo , Unión Proteica/efectos de los fármacos , Pirazoles/química , Pirazoles/farmacología , Estabilidad del ARN/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Xanthomonas/efectos de los fármacos , Xanthomonas/fisiologíaRESUMEN
We have previously shown that 5' halves from tRNAGlyGCC and tRNAGluCUC are the most enriched small RNAs in the extracellular space of human cell lines, and especially in the non-vesicular fraction. Extracellular RNAs are believed to require protection by either encapsulation in vesicles or ribonucleoprotein complex formation. However, deproteinization of non-vesicular tRNA halves does not affect their retention in size-exclusion chromatography. Thus, we considered alternative explanations for their extracellular stability. In-silico analysis of the sequence of these tRNA-derived fragments showed that tRNAGly 5' halves can form homodimers or heterodimers with tRNAGlu 5' halves. This capacity is virtually unique to glycine tRNAs. By analyzing synthetic oligonucleotides by size exclusion chromatography, we provide evidence that dimerization is possible in vitro. tRNA halves with single point substitutions preventing dimerization are degraded faster both in controlled nuclease digestion assays and after transfection in cells, showing that dimerization can stabilize tRNA halves against the action of cellular nucleases. Finally, we give evidence supporting dimerization of endogenous tRNAGlyGCC 5' halves inside cells. Considering recent reports have shown that 5' tRNA halves from Ala and Cys can form tetramers, our results highlight RNA intermolecular structures as a new layer of complexity in the biology of tRNA-derived fragments.
Asunto(s)
Dimerización , Estabilidad del ARN , ARN de Transferencia de Ácido Glutámico/metabolismo , ARN de Transferencia de Glicerina/metabolismo , Ribonucleasas/metabolismo , Región de Flanqueo 5' , Secuencia de Bases , Ácido Glutámico/metabolismo , Glicina/metabolismo , Humanos , Células MCF-7 , Conformación de Ácido Nucleico , ARN de Transferencia de Ácido Glutámico/química , ARN de Transferencia de Glicerina/químicaRESUMEN
Tuberculosis is a disease caused by Mycobacterium tuberculosis (Mtb). Innate immunity is the first line of defense against Mtb and malfunctions in any of its components are associated with the susceptibility to the disease. Epithelial products such as host defense peptides (HDPs) are the first molecules produced to counteract the infection. Although a wide variety of HDPs are produced by epithelial cells only a few of them have been studied during Mtb infection. Here, we assessed the expression and production of the HDPs psoriasin, secreted phospholipases A2 (sPLA2-IIA) and Ribonuclease (RNase) 7 in airway epithelial cells (NCI-H292), type II pneumocytes (A549 cells) and monocyte-derived macrophages from human peripheral blood mononuclear cells and from the human cell line THP1 after Mtb in vitro infection. Results show that psoriasin and sPLA2-IIA were not induced by Mtb in any of the evaluated cells, while RNase 7 was overexpressed in infected airway epithelial cells. Intracellular analysis by flow cytometry demonstrated that the highest levels of RNase 7 were observed 6 h post-infection and the induction was dependent on direct interaction between airway epithelial cells and Mtb. In addition, analysis by electron microscopy showed that RNase 7 was capable of attaching to the cell wall of intracellular mycobacteria. Our studies suggest that the induction of RNase 7 in response to Mtb could have a role in anti-mycobacterial immunity, which needs to be studied as an innate immune mechanism.
Asunto(s)
Células Epiteliales Alveolares/microbiología , Fosfolipasas A2 Grupo II/metabolismo , Interacciones Huésped-Patógeno , Mycobacterium tuberculosis/metabolismo , Ribonucleasas/metabolismo , Proteína A7 de Unión a Calcio de la Familia S100/metabolismo , Células A549 , Células Epiteliales Alveolares/inmunología , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Monocitos/inmunología , Monocitos/microbiologíaRESUMEN
KEY MESSAGE: The manuscript by Alves et al. entitled "Genome-wide identification and characterization of tRNA-derived RNA fragments in land plants" describes the identification and characterization of tRNAderived sRNA fragments in plants. By combining bioinformatic analysis and genetic and molecular approaches, we show that tRF biogenesis does not rely on canonical microRNA/siRNA processing machinery (i.e., independent of DICER-LIKE proteins). Moreover, we provide evidences that the Arabidopsis S-like Ribonuclease 1 (RNS1) might be involved in the biogenesis of tRFs. Detailed analyses showed that plant tRFs are sorted into different types of ARGONAUTE proteins and that they have potential target candidate genes. Our work advances the understanding of the tRF biology in plants by providing evidences that plant and animal tRFs shared common features and raising the hypothesis that an interplay between tRFs and other sRNAs might be important to fine-tune gene expression and protein biosynthesis in plant cells. Small RNA (sRNA) fragments derived from tRNAs (3'-loop, 5'-loop, anti-codon loop), named tRFs, have been reported in several organisms, including humans and plants. Although they may interfere with gene expression, their biogenesis and biological functions in plants remain poorly understood. Here, we capitalized on small RNA sequencing data from distinct species such as Arabidopsis thaliana, Oryza sativa, and Physcomitrella patens to examine the diversity of plant tRFs and provide insight into their properties. In silico analyzes of 19 to 25-nt tRFs derived from 5' (tRF-5s) and 3'CCA (tRF-3s) tRNA loops in these three evolutionary distant species showed that they are conserved and their abundance did not correlate with the number of genomic copies of the parental tRNAs. Moreover, tRF-5 is the most abundant variant in all three species. In silico and in vivo expression analyses unraveled differential accumulation of tRFs in Arabidopsis tissues/organs, suggesting that they are not byproducts of tRNA degradation. We also verified that the biogenesis of most Arabidopsis 19-25 nt tRF-5s and tRF-3s is not primarily dependent on DICER-LIKE proteins, though they seem to be associated with ARGONAUTE proteins and have few potential targets. Finally, we provide evidence that Arabidopsis ribonuclease RNS1 might be involved in the processing and/or degradation of tRFs. Our data support the notion that an interplay between tRFs and other sRNAs might be important to fine tune gene expression and protein biosynthesis in plant cells.
Asunto(s)
Genoma de Planta , ARN de Planta/química , ARN de Transferencia/química , Arabidopsis/genética , Arabidopsis/metabolismo , Bryopsida/genética , Bryopsida/metabolismo , Biología Computacional , Oryza/genética , Oryza/metabolismo , Estrés Oxidativo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiología , ARN de Planta/metabolismo , ARN de Transferencia/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Ribonucleasas/genética , Ribonucleasas/metabolismo , Ribonucleasas/fisiologíaRESUMEN
Con el objetivo de aislar y caracterizar parcialmente las enzimas ribonucleasas (RNasas) contenidas en el látex de Calotropis procera y Pedilanthus tithymaloides, se colectaron muestras de plantas adultas. Las proteínas solubles fueron extraídas con acetato de sodio y centrifugación a 16.000 x g durante 15 min y fraccionadas por cromatografía de intercambio iónico. Se estimó la masa molecular a través de ecuaciones de regresión lineal. Se realizaron pruebas de glicosilación. En ambas especies, las proteínas con actividad RNasa presentaron una masa molecular entre 28 y 30 kDa. No existe evidencia de proteínas glicosiladas en el látex de C. procera. En P. tithymaloides la RNasa es una proteína glicosilada.
In order to isolate and characterize partially ribonucleases (RNases) enzymes contained in the latex from Calotropis procera and Pedilanthus tithymaloides, samples were collected from mature plants. Soluble proteins were extracted with sodium acetate and centrifugation at 16,000 xg for 15 min and fractionated by ion exchange chromatography. Molecular mass was estimated by linear regression equations. Glycosylation tests were conducted. In both species, proteins with RNase activity showed a molecular mass between 28 and 30 kDa. No evidence of glycosylated proteins in latex from C. procera. In P. tithymaloides, RNase may be a glycosylated protein.
Asunto(s)
Calotropis/enzimología , Euphorbiaceae/enzimología , Látex/química , Ribonucleasas/aislamiento & purificación , Ribonucleasas/metabolismo , Calotropis/química , Euphorbiaceae/química , GlicosilaciónRESUMEN
A combined strategy of phosphate (Pi) remobilization from internal and external RNA sources seems to be conserved in plants exposed to Pi starvation. Thus far, the only ribonucleases (RNases) reported to be induced in Nicotiana alata undergoing Pi deprivation are extracellular S-like RNase NE and NnSR1. NnSR1 is a class III non S-RNase of unknown subcellular location. Here, we examine the hypothesis that NnSR1 is an intracellular RNase derived from the self-incompatibility system with specific expression in self-incompatible Nicotiana alata. NnSR1 was not induced in self-compatible Nicotiana species exposed to Pi deprivation. NnSR1 conjugated with a fluorescent protein and transiently expressed in Arabidopsis protoplasts and Nicotiana leaves showed that the fusion protein co-localized with an endoplasmic reticulum (ER) marker. Subcellular fractionation by ultracentrifugation of roots exposed to Pi deprivation revealed that the native NnSR1 migrated in parallel with the BiP protein, a typical ER marker. To our knowledge, NnSR1 is the first class III RNase reported to be localized in ER compartments. The induction of NnSR1 was detected earlier than the extracellular RNase NE, suggesting that intracellular RNA may be the first source of Pi used by the cell under Pi stress.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Nicotiana/genética , Proteínas de Plantas/genética , Ribonucleasas/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Retículo Endoplásmico/metabolismo , Datos de Secuencia Molecular , Fosfatos/deficiencia , Filogenia , Hojas de la Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Protoplastos/metabolismo , Ribonucleasas/química , Ribonucleasas/metabolismo , Alineación de Secuencia , Nicotiana/enzimologíaRESUMEN
RNA metabolism is a critical but frequently overlooked control element affecting virtually every cellular process in bacteria. RNA processing and degradation is mediated by a suite of ribonucleases having distinct cleavage and substrate specificity. Here, we probe the role of two ribonucleases (RNase III and RNase J) in the emerging model system Streptomyces venezuelae. We show that each enzyme makes a unique contribution to the growth and development of S. venezuelae and further affects the secondary metabolism and antibiotic production of this bacterium. We demonstrate a connection between the action of these ribonucleases and translation, with both enzymes being required for the formation of functional ribosomes. RNase III mutants in particular fail to properly process 23S rRNA, form fewer 70S ribosomes, and show reduced translational processivity. The loss of either RNase III or RNase J additionally led to the appearance of a new ribosomal species (the 100S ribosome dimer) during exponential growth and dramatically sensitized these mutants to a range of antibiotics.
Asunto(s)
Antibacterianos/biosíntesis , Ribonucleasas/metabolismo , Ribosomas/metabolismo , Streptomyces/enzimología , Eliminación de Gen , Biosíntesis de Proteínas , Ribonucleasas/genética , Metabolismo Secundario , Streptomyces/genética , Streptomyces/crecimiento & desarrolloRESUMEN
PURPOSE: To obtain a decellularized tracheal scaffold associating traditional approaches with the novel light-emitting diode (LED) proposal. METHODS: This study was performed with New Zealand adult rabbits weighing 3.0 - 4.0 kg. Different protocols (22) were used combining physical (agitation and LED irradiation), chemical (SDS and Triton X-100 detergents), and enzymatic methods (DNase and RNase). RESULTS: Generally, the cells surrounding soft tissues were successfully removed, but none protocol removed cells from the tracheal cartilage. However, longer protocols were more effective. The cost-benefits relation of the enzymatic processes was not favorable. It was possible to find out that the cartilaginous tissue submitted to the irradiation with LED 630nm and 475 nm showed an increased number of gaps without cells, but several cells were observed to be still present. CONCLUSION: The light-emitting diode is a promising tool for decellularization of soft tissues.
Asunto(s)
Luz , Ingeniería de Tejidos/métodos , Andamios del Tejido , Tráquea/ultraestructura , Animales , Desoxirribonucleasas/metabolismo , Detergentes/farmacología , Matriz Extracelular/ultraestructura , Conejos , Ribonucleasas/metabolismo , Tráquea/efectos de los fármacos , Tráquea/enzimologíaRESUMEN
PURPOSE: To obtain a decellularized tracheal scaffold associating traditional approaches with the novel light-emitting diode (LED) proposal. METHODS: This study was performed with New Zealand adult rabbits weighing 3.0 - 4.0 kg. Different protocols (22) were used combining physical (agitation and LED irradiation), chemical (SDS and Triton X-100 detergents), and enzymatic methods (DNase and RNase). RESULTS: Generally, the cells surrounding soft tissues were successfully removed, but none protocol removed cells from the tracheal cartilage. However, longer protocols were more effective. The cost-benefits relation of the enzymatic processes was not favorable. It was possible to find out that the cartilaginous tissue submitted to the irradiation with LED 630nm and 475 nm showed an increased number of gaps without cells, but several cells were observed to be still present. CONCLUSION: The light-emitting diode is a promising tool for decellularization of soft tissues. .
Asunto(s)
Animales , Conejos , Luz , Andamios del Tejido , Ingeniería de Tejidos/métodos , Tráquea/ultraestructura , Desoxirribonucleasas/metabolismo , Detergentes/farmacología , Matriz Extracelular/ultraestructura , Ribonucleasas/metabolismo , Tráquea/efectos de los fármacos , Tráquea/enzimologíaRESUMEN
The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation.