RESUMO
Inflammatory cytokines are key signaling molecules that can promote an immune response, thus their RNA turnover must be tightly controlled during infection. Most studies investigate the RNA decay pathways in the cytosol or nucleoplasm but never focused on the nucleolus. Although this organelle has well-studied roles in ribosome biogenesis and cellular stress sensing, the mechanism of RNA decay within the nucleolus is not completely understood. Here, we report that the nucleolus is an essential site of inflammatory pre-mRNA instability during infection. RNA-sequencing analysis reveals that not only do inflammatory genes have higher intronic read densities compared with non-inflammatory genes, but their pre-mRNAs are highly enriched in nucleoli during infection. Notably, nucleolin (NCL) acts as a guide factor for recruiting cytosine or uracil (C/U)-rich sequence-containing inflammatory pre-mRNAs and the Rrp6-exosome complex to the nucleolus through a physical interaction, thereby enabling targeted RNA delivery to Rrp6-exosomes and subsequent degradation. Consequently, Ncl depletion causes aberrant hyperinflammation, resulting in a severe lethality in response to LPS. Importantly, the dynamics of NCL post-translational modifications determine its functional activity in phases of LPS. This process represents a nucleolus-dependent pathway for maintaining inflammatory gene expression integrity and immunological homeostasis during infection.
Assuntos
Nucléolo Celular , Lipopolissacarídeos , Nucléolo Celular/metabolismo , Núcleo Celular , Lipopolissacarídeos/metabolismo , RNA/metabolismo , Estabilidade de RNARESUMO
The core plant microprocessor consists of DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1) and plays a pivotal role in microRNA (miRNA) biogenesis. However, the proteolytic regulation of each component remains elusive. Here, we show that HYL1-CLEAVAGE SUBTILASE 1 (HCS1) is a cytoplasmic protease for HYL1-destabilization. HCS1-excessiveness reduces HYL1 that disrupts miRNA biogenesis, while HCS1-deficiency accumulates HYL1. Consistently, we identified the HYL1K154A mutant that is insensitive to the proteolytic activity of HCS1, confirming the importance of HCS1 in HYL1 proteostasis. Moreover, HCS1-activity is regulated by light/dark transition. Under light, cytoplasmic CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase suppresses HCS1-activity. COP1 sterically inhibits HCS1 by obstructing HYL1 access into the catalytic sites of HCS1. In contrast, darkness unshackles HCS1-activity for HYL1-destabilization due to nuclear COP1 relocation. Overall, the COP1-HYL1-HCS1 network may integrate two essential cellular pathways: the miRNA-biogenetic pathway and light signaling pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Folhas de Planta/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The shift of dark-grown seedlings into light causes enormous transcriptome changes followed by a dramatic developmental transition. Here, we show that microRNA (miRNA) biogenesis also undergoes regulatory changes during de-etiolation. Etiolated seedlings maintain low levels of primary miRNAs (pri-miRNAs) and miRNA processing core proteins, such as Dicer-like 1, SERRATE, and HYPONASTIC LEAVES 1, whereas during de-etiolation both pri-miRNAs and the processing components accumulate to high levels. However, the levels of most miRNAs do not notably increase in response to light. To reconcile this inconsistency, we demonstrated that an unknown suppressor decreases miRNA-processing activity and light-induced SMALL RNA DEGRADING NUCLEASE 1 shortens the half-life of several miRNAs in de-etiolated seedlings. Taken together, these data suggest a novel mechanism, miRNA-biogenetic inconsistency, which accounts for the intricacy of miRNA biogenesis during de-etiolation. This mechanism is essential for the survival of de-etiolated seedlings after long-term skotomorphogenesis and their optimal adaptation to ever-changing light conditions.
Assuntos
Arabidopsis/genética , Arabidopsis/efeitos da radiação , Luz , MicroRNAs/biossíntese , Plântula/fisiologia , Plântula/efeitos da radiação , Arabidopsis/fisiologia , Transcriptoma/efeitos da radiação , Regulação para Cima/efeitos da radiaçãoRESUMO
BACKGROUND: Controlled turnover of proteins as mediated by the ubiquitin proteasome system (UPS) is an important element in plant defense against environmental and pathogen stresses. E3 ligases play a central role in subjecting proteins to hydrolysis by the UPS. Recently, it has been demonstrated that a specific class of E3 ligases termed the U-box ligases are directly associated with the defense mechanisms against abiotic and biotic stresses in several plants. However, no studies on U-box E3 ligases have been performed in one of the important staple crops, barley. RESULTS: In this study, we identified 67 putative U-box E3 ligases from the barley genome and expressed sequence tags (ESTs). Similar to Arabidopsis and rice U-box E3 ligases, most of barley U-box E3 ligases possess evolutionary well-conserved domain organizations. Based on the domain compositions and arrangements, the barley U-box proteins were classified into eight different classes. Along with this new classification, we refined the previously reported classifications of U-box E3 ligase genes in Arabidopsis and rice. Furthermore, we investigated the expression profile of 67 U-box E3 ligase genes in response to drought stress and pathogen infection. We observed that many U-box E3 ligase genes were specifically up-and-down regulated by drought stress or by fungal infection, implying their possible roles of some U-box E3 ligase genes in the stress responses. CONCLUSION: This study reports the classification of U-box E3 ligases in barley and their expression profiles against drought stress and pathogen infection. Therefore, the classification and expression profiling of barley U-box genes can be used as a platform to functionally define the stress-related E3 ligases in barley.
Assuntos
Regulação da Expressão Gênica de Plantas , Hordeum/genética , Interações Hospedeiro-Parasita/genética , Proteínas de Plantas/genética , Ubiquitina-Proteína Ligases/genética , Sequência de Aminoácidos , Arabidopsis/genética , Ascomicetos/patogenicidade , Secas , Genoma de Planta , Hordeum/crescimento & desenvolvimento , Oryza/genética , Filogenia , Proteínas de Plantas/classificação , Plântula/microbiologia , Alinhamento de Sequência , Ubiquitina-Proteína Ligases/classificaçãoRESUMO
MISFOLDED PROTEIN SENSING RING1 (MPSR1) is a chaperone-independent E3 ubiquitin ligase that participates in protein quality control by eliminating misfolded proteins in Arabidopsis (Arabidopsis thaliana). Here, we report that in the early stages of proteotoxic stress, cellular levels of MPSR1 increased immediately, whereas levels of HEAT SHOCK PROTEIN90.1 (AtHSP90.1) were unaltered despite massively upregulated transcription. At this stage, the gene-silencing pathway mediated by microRNA 414 (miR414) suppressed AtHSP90.1 translation. By contrast, under prolonged stress, AtHSP90.1 was not suppressed, and instead competed with MPSR1 to act on misfolded proteins, promoting the destruction of MPSR1. Deficiency or excess of MPSR1 significantly abolished or intensified the suppression of AtHSP90.1, respectively. Similar to the MPSR1-overexpressing transgenic plants, the miR414-overexpressing plants showed an increased tolerance to proteotoxic stress as compared to the wild-type plants. Although the functional relationship between MPSR1 and miR414 remains unclear, both MPSR1 and miR414 demonstrated negative modulation of the expression of AtHSP90.1. The inverse correlation between MPSR1 and AtHSP90.1 via miR414 may adjust the set-point of the HSP90-mediated protein quality control process in response to increasing stress intensity in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citoplasma/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Proteínas de Choque Térmico HSP90/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Dobramento de Proteína , Estresse Fisiológico/genética , Transcriptoma/genéticaRESUMO
The scaffolding DNA sequence and the size of silver nanoclusters (AgNCs), confined in a DNA template are the key parameters in determining the fluorescent properties of DNA-stabilized silver nanoclusters (DNA/AgNCs). In addition, we suggest here that the structural shift of a DNA hairpin-dimer is as important as the DNA sequence in determining the emission wavelength of DNA/AgNCs. Furthermore, we show that the structural shift post AgNC formation can be triggered by incubation time and pre-AgNC formation under salt conditions. As an important factor in predicting the emission properties of DNA/AgNCs, the modulation of DNA secondary structures with either sequence changes or ionic conditions can be applied for the dual-color detection system of a target molecule. Particularly, the dual-color detection method may increase the reliability of DNA/AgNC sensors for miRNAs.
Assuntos
DNA/química , Nanopartículas Metálicas/química , Prata/química , Sequência de Bases , Técnicas Biossensoriais , Dimerização , MicroRNAs/análise , Conformação de Ácido Nucleico , Espectrometria de FluorescênciaRESUMO
Light is the most influential environmental stimulus for plant growth. In response to deficient light, plants reprogram their development to adjust their growth in search for a light source. A fine reprogramming of gene expression orchestrates this adaptive trait. Here we show that plants alter microRNA (miRNA) biogenesis in response to light transition. When plants suffer an unusual extended period of light deprivation, the miRNA biogenesis factor HYPONASTIC LEAVES 1 (HYL1) is degraded but an inactive pool of phosphorylated protein remains stable inside the nucleus. Degradation of HYL1 leads to the release of gene silencing, triggering a proper response to dark and shade. Upon light restoration, a quick dephosphorylation of HYL1 leads to the reactivation of miRNA biogenesis and a switch toward a developmental program that maximizes the light uptake. Our findings define a unique and fast regulatory mechanism controlling the plant silencing machinery during plant light response.
Assuntos
Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Inativação Gênica , Luz , MicroRNAs/genética , Mutação , Fosforilação , Folhas de Planta/metabolismo , Processamento Pós-Transcricional do RNA/fisiologiaRESUMO
KEY MESSAGE: BPH1 acts as a substrate receptor of CRL3 complex and negatively regulates ABA-mediated cellular responses. The study on its function provides information that helps further understand the relationship between ABA signaling and UPS. Abscisic acid (ABA) plays a crucial role in a variety of cellular processes, including seed dormancy, inhibition of seedling growth, and drought resistance in plants. Cullin3-RING E3 ligase (CRL3) complex is a type of multi-subunit E3 ligase, and BTB/POZ protein, a component of CRL3 complex, functions as a receptor to determine a specific substrate. To elucidate the CRL3 complex that participates in ABA-mediated cellular processes, we first investigated ABA-inducible BTB/POZ genes based on data from the AtGenExpress Visualization Tool (AVT). We then isolated an ABA-inducible gene encoding a potential CRL3 substrate receptor in Arabidopsis, BPH1 (BTB/POZ protein hypersensitive to ABA 1). The isolate gene has a BTB/POZ domain and a NPH3 domain within its N-terminal and C-terminal region, respectively. Yeast two-hybrid and co-immunoprecipitation assays showed that BPH1 physically interacted with cullin3a, a scaffold protein of CRL3, suggesting that it functions as an Arabidopsis CRL3 substrate receptor. The functional mutation of BPH1 caused delayed seed germination in response to ABA and enhanced sensitivity by NaCl and mannitol treatments as ABA-related stresses. Moreover, bph1 mutants exhibited enhanced stomatal closure under ABA application and reduced water loss when compared with wild-type, implying their enhanced tolerance to drought stress. Based on the information from microarray/AVT data and expression analysis of various ABA-inducible genes between wild-type and bph1 plants following ABA treatments, we concluded loss of BPH1 resulted in hyper-induction of a large portion of ABA-inducible genes in response to ABA. Taken together, these results show that BPH1 is negatively involved in ABA-mediated cellular events.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ubiquitina-Proteína Ligases/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação/efeitos dos fármacos , Germinação/genética , Mutação , Filogenia , Reguladores de Crescimento de Plantas/farmacologia , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/genética , Estômatos de Plantas/metabolismo , Ligação Proteica , Sementes/genética , Sementes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Cloreto de Sódio/farmacologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: The E3 ubiquitin ligase constitutive photomorphogenic 1 (COP1) mediates cell survival, growth, and development, and interacts with the tumor suppressor protein p53 to induce its ubiquitination and degradation. Recent studies reported that COP1 overexpression is associated with increased cell proliferation, transformation, and disease progression in a variety of cancer types. In this study, we investigated whether COP1 regulates p53-mediated cell cycle arrest and apoptosis in human breast cancer cell lines. METHODS: We downregulated COP1 expression using lentiviral particles expressing short hairpin RNA (shRNA) targeting COP1 and measured the effects of the knockdown in three different breast cancer cell lines. RESULTS: COP1 silencing resulted in p53 activation, which induced the expression of p21 and p53-upregulated modulator of apoptosis (PUMA) expression, and reduced the levels of cyclin-dependent kinase 2 (CDK2). Notably, knockdown of COP1 was associated with cell cycle arrest during the G0/G1 phase. CONCLUSIONS: The COP1-mediated degradation of p53 regulates cancer cell growth and apoptosis. Our results indicate that COP1 regulates human breast cancer cell proliferation and apoptosis in a p53-dependent manner. These findings suggest that COP1 might be a promising potential target for breast cancer-related gene therapy.
Assuntos
Apoptose/fisiologia , Neoplasias da Mama/patologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estabilidade Proteica , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Cyanogenic glucosides are present in many plants, including eudicots, monocots, and ferns and function as defence compounds based on their ability to release hydrogen cyanide. In this study, the diurnal rhythm of cyanogenic glucoside content and of transcripts and enzymes involved in their biosynthesis was monitored in cassava plants grown in a glasshouse under natural light conditions. Transcripts of CYP79D1, CYP79D2, CYP71E7/11, and UGT85K5 were at minimal levels around 9 p.m., increased during the night and decreased following onset of early morning light. Transcripts of UGT85K4 and HNL10 showed more subtle variations with a maximum reached in the afternoon. Western blots showed that the protein levels of CYP71E7/11 and UGT85K4/5 decreased during the light period to a near absence around 4 p.m. and then recovered during the dark period. Transcript and protein levels of linamarase were stable throughout the 24-hr cycle. The linamarin content increased during the dark period. In the light period, spikes in the incoming solar radiation were found to result in concomitantly reduced linamarin levels. In silico studies of the promoter regions of the biosynthetic genes revealed a high frequency of light, abiotic stress, and development-related transcription factor binding motifs. The synthesis and endogenous turnover of linamarin are controlled both at the transcript and protein levels. The observed endogenous turnover of linamarin in the light period may offer a source of reduced nitrogen to balance photosynthetic carbon fixation. The rapid decrease in linamarin content following light spikes suggests an additional function of linamarin as a ROS scavenger.
RESUMO
Ubiquitin E3 ligases are crucial for eliminating misfolded proteins before they form cytotoxic aggregates that threaten cell fitness and survival. However, it remains unclear how emerging misfolded proteins in the cytoplasm can be selectively recognized and eliminated by E3 ligases in plants. We found that Misfolded Protein Sensing RING E3 ligase 1 (MPSR1) is an indispensable E3 ligase required for plant survival after protein-damaging stress. Under no stress, MPSR1 is prone to rapid degradation by the 26S proteasome, concealing its protein quality control (PQC) E3 ligase activity. Upon proteotoxic stress, MPSR1 directly senses incipient misfolded proteins and tethers ubiquitins for subsequent degradation. Furthermore, MPSR1 sustains the structural integrity of the proteasome complex at the initial stage of proteotoxic stress. Here, we suggest that the MPSR1 pathway is a constitutive mechanism for proteostasis under protein-damaging stress, as a front-line surveillance system in the cytoplasm.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Dobramento de Proteína , Proteostase , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Citoplasma/metabolismo , DNA de Plantas , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes , Análise de Sequência , Análise de Sequência de RNA , Estresse Psicológico , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/metabolismo , Leveduras/genéticaRESUMO
Plants are constantly exposed to a variety of abiotic stresses, such as drought, heat, cold, flood, and salinity. To survive under such unfavorable conditions, plants have evolutionarily developed their own resistant-mechanisms. For several decades, many studies have clarified specific stress response pathways of plants through various molecular and genetic studies. In particular, it was recently discovered that ubiquitin proteasome system (UPS), a regulatory mechanism for protein turn over, is greatly involved in the stress responsive pathways. In the UPS, many E3 ligases play key roles in recognizing and tethering poly-ubiquitins on target proteins for subsequent degradation by the 26S proteasome. Here we discuss the roles of RING ligases that have been defined in related to abiotic stress responses in plants. [BMB Reports 2017; 50(8): 393-400].
Assuntos
Plantas/genética , Plantas/metabolismo , Estresse Fisiológico/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
AtAIRP2 is a cytosolic RING-type E3 ubiquitin ligase that positively regulates an abscisic acid (ABA) response in Arabidopsis (Arabidopsis thaliana). Yeast two-hybrid screening using AtAIRP2 as bait identified ATP1 (AtAIRP2 Target Protein1) as a substrate of AtAIRP2. ATP1 was found to be identical to SDIRIP1, which was reported recently to be a negative factor in ABA signaling and a target protein of the RING E3 ligase SDIR1. Accordingly, ATP1 was renamed ATP1/SDIRIP1. A specific interaction between AtAIRP2 and ATP1/SDIRIP1 and ubiquitination of ATP1/SDIRIP1 by AtAIRP2 were demonstrated in vitro and in planta. The turnover of ATP1/SDIRIP1 was regulated by AtAIRP2 in cell-free degradation and protoplast cotransfection assays. The ABA-mediated germination assay of 35S:ATP1/SDIRIP1-RNAi/atairp2 double mutant progeny revealed that ATP1/SDIRIP1 acts downstream of AtAIRP2. AtAIRP2 and SDIR1 reciprocally complemented the ABA- and salt-insensitive germination phenotypes of sdir1 and atairp2 mutants, respectively, indicating their combinatory roles in seed germination. Subcellular localization and bimolecular fluorescence complementation experiments in the presence of MG132, a 26S proteasome inhibitor, showed that AtAIRP2 and ATP1/SDIRIP1 were colocalized to the cytosolic spherical body, which lies in close proximity to the nucleus, in tobacco (Nicotiana benthamiana) leaf cells. The 26S proteasome subunits RPN12a and RPT1 and the molecular chaperones HSP70 and HSP101 were colocalized to these discrete punctae-like structures. These results raised the possibility that AtAIRP2 and ATP1/SDIRIP1 interact in the cytosolic spherical compartment. Collectively, our data suggest that the down-regulation of ATP1/SDIRIP1 by AtAIRP2 and SDIR1 RING E3 ubiquitin ligases is critical for ABA and high-salinity responses during germination in Arabidopsis.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Salinidade , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Compartimento Celular , Citosol/efeitos dos fármacos , Citosol/metabolismo , Regulação para Baixo/genética , Epistasia Genética/efeitos dos fármacos , Teste de Complementação Genética , Germinação/efeitos dos fármacos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Epiderme Vegetal/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Frações Subcelulares/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Nicotiana/citologiaRESUMO
A highly coordinated complex known as the microprocessor precisely processes primary transcripts of MIRNA genes into mature miRNAs. In plants, the microprocessor minimally consists of three components: Dicer-like protein 1 (DCL1), HYPONASTIC LEAF 1 (HYL1), and SERRATE (SE). To precisely modulate miRNA maturation, the microprocessor cooperates with at least 12 proteins in plants. In addition, we here show the involvement of a novel gene, HYL1-interacting GIY-YIG-like endonuclease (HIGLE). The encoded protein has a GIY-YIG domain that is generally found within a class of homing endonucleases. HIGLE directly interacts with the microprocessor components HYL1 and SE. Unlike the functions of other GIY-YIG endonucleases, the catalytic core of HIGLE has both DNase and RNase activities that sufficiently processes miRNA precursors into short fragments in vitro.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Endorribonucleases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Domínio Catalítico , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Endonucleases/química , Endonucleases/genética , Endorribonucleases/química , Endorribonucleases/genética , MicroRNAs/metabolismo , Filogenia , Domínios Proteicos , Técnicas do Sistema de Duplo-HíbridoRESUMO
MicroRNAs (miRNAs) are essential small RNA molecules (20-24 nt) that negatively regulate the expression of target genes at the post-transcriptional level. Due to their roles in a variety of biological processes, the aberrant expression profiles of miRNAs have been identified as biomarkers for many diseases, such as cancer, diabetes, cardiovascular disease and neurodegenerative diseases. In order to precisely, rapidly and economically monitor the expression of miRNAs, many cutting-edge nanotechnologies have been developed. One of the nanotechnologies, based on DNA encapsulated silver nanoclusters (DNA/AgNCs), has increasingly been adopted to create nanoscale bio-sensing systems due to its attractive optical properties, such as brightness, tuneable emission wavelengths and photostability. Using the DNA/AgNCs sensor methods, the presence of miRNAs can be detected simply by monitoring the fluorescence alteration of DNA/AgNCs sensors. We introduce these DNA/ AgNCs sensor methods and discuss their possible applications for detecting miRNA biomarkers in neurodegenerative diseases.
RESUMO
A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-ZIP) transcription factors are key mediators in the regulation of adaxial-abaxial patterning. Their expression is restricted adaxially during early development by the abaxially expressed microRNA (MIR)165/166, yet the mechanism that restricts MIR165/166 expression to abaxial leaf tissues remains unknown. Here, we show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets.
Assuntos
Proteínas de Homeodomínio/genética , Zíper de Leucina/genética , MicroRNAs/genética , Desenvolvimento Vegetal/genética , Folhas de Planta/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Biomarcadores , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Modelos Biológicos , Ligação Proteica , Característica Quantitativa Herdável , Elementos de RespostaRESUMO
Post-translational modifications (PTMs) of proteins are essential to increase the functional diversity of the proteome. By adding chemical groups to proteins, or degrading entire proteins by phosphorylation, glycosylation, ubiquitination, neddylation, acetylation, lipidation, and proteolysis, the complexity of the proteome increases, and this then influences most biological processes. Although small RNAs are crucial regulatory elements for gene expression in most eukaryotes, PTMs of small RNA microprocessor and RNA silencing components have not been extensively investigated in plants. To date, several studies have shown that the proteolytic regulation of AGOs is important for host-pathogen interactions. DRB4 is regulated by the ubiquitin-proteasome system, and the degradation of HYL1 is modulated by a de-etiolation repressor, COP1, and an unknown cytoplasmic protease. Here, we discuss current findings on the PTMs of microprocessor and RNA silencing components in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , Plantas , Proteínas de Ligação a RNA/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Argonautas/genética , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , MicroRNAs/genética , Processamento de Proteína Pós-Traducional , Proteólise , Interferência de RNA , Proteínas de Ligação a RNA/genética , Ubiquitina/metabolismoRESUMO
MicroRNAs (miRNAs), small non-coding RNA molecules, are important biomarkers for research and medical purposes. Here, we describe the development of a fast and simple method using highly fluorescent oligonucleotide-silver nanocluster probes (DNA/AgNCs) to efficiently detect specific miRNAs. Due to the great sequence diversity of miRNAs in humans and other organisms, a uniform strategy for miRNA detection is attractive. The concept presented is an oligonucleotide-based locking-to-unlocking system that can be endowed with miRNA complementarity while maintaining the same secondary structure. The locking-to-unlocking system is based on fold-back anchored DNA templates that consist of a cytosine-rich loop for AgNCs stabilization, an miRNA recognition site and an overlap region for hairpin stabilization. When an miRNA is recognized, fluorescence in the visible region is specifically extinguished in a concentration-dependent manner. Here, the exact composition of the fold-back anchor for the locking-to-unlocking system has been systematically optimized, balancing propensity for loop-structure formation, encapsulation of emissive AgNCs and target sensitivity. It is demonstrated that the applied strategy successfully can detect a number of cancer related miRNAs in RNA extracts from human cancer cell lines.
Assuntos
Corantes Fluorescentes/química , Nanopartículas Metálicas/química , MicroRNAs/análise , Sondas de Oligonucleotídeos/química , RNA Neoplásico/análise , Pareamento de Bases , Sequência de Bases , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Citosina/química , Corantes Fluorescentes/síntese química , Humanos , Nanopartículas Metálicas/ultraestrutura , MicroRNAs/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos/síntese química , RNA Neoplásico/metabolismo , Prata/química , Espectrometria de FluorescênciaRESUMO
Plants have developed a variety of complicated responses to cope with drought, one of the most challenging environmental stresses. As a quick response, plants rapidly inhibit stomatal opening under the control of abscisic acid (ABA) signaling pathway, in order to preserve water. Here, we report that Arabidopsis Tóxicos en Levadura (ATL), a RING-type E3 ubiquitin ligase, mediates the ABA-dependent stomatal closure. In contrast to wild-type plants, the stomatal closure was fully impaired in atatl78 mutant plants even in the presence of exogenous ABA and reactive oxygen species (ROS). Besides, under high concentrations of Ca(2+), a down-stream signaling molecule of ABA signaling pathway, atatl78 mutant plants successfully closed the pores. Furthermore, AtATL78 protein indirectly associated with catalases and the deficiency of AtATL78 led the reduction of catalase activity and H2O2, implying the function of AtATL78 in the modulation of ROS activity. Based on these results, we suggest that AtATL78 possibly plays a role in promoting ROS-mediated ABA signaling pathway during drought stress.
Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/fisiologia , Secas , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Adaptação Fisiológica/fisiologiaRESUMO
Drought stress strongly affects plant growth and development, directly connected with crop yields, accordingly. However, related to the function of U-BOX E3 ligases, the underlying molecular mechanisms of desiccation stress response in plants are still largely unknown. Here we report that PUB22 and PUB23, two U-box E3 ligase homologs, tether ubiquitins to 19S proteasome regulatory particle (RP) subunit RPN6, leading to its degradation. RPN6 was identified as an interacting substrate of PUB22 by yeast two-hybrid screening, and in vitro pull-down assay confirmed that RPN6 interacts not only with PUB22, but also with PUB23. Both PUB22 and PUB23 were able to conjugate ubiquitins on RPN6 in vitro. Furthermore, RPN6 showed a shorter protein half-life in PUB22 overexpressing plants than in wild-type, besides RPN6 was significantly stabilized in pub22pub23 double knockout plants. Taken together, these results solidify a notion that PUB22 and PUB23 can alter the activity of 26S proteasome in response to drought stress.