RESUMEN
N6-methyladenosine (m6A) modifications play crucial roles in RNA metabolism. How m6A regulates RNA polymerase II (RNA Pol II) transcription remains unclear. We find that 7SK small nuclear RNA (snRNA), a regulator of RNA Pol II promoter-proximal pausing, is highly m6A-modified in non-small cell lung cancer (NSCLC) cells. In A549 cells, we identified eight m6A sites on 7SK and discovered methyltransferase-like 3 (METTL3) and alkB homolog 5 (ALKBH5) as the responsible writer and eraser. When the m6A-7SK is specifically erased by a dCasRx-ALKBH5 fusion protein, A549 cell growth is attenuated due to reduction of RNA Pol II transcription. Mechanistically, removal of m6A leads to 7SK structural rearrangements that facilitate sequestration of the positive transcription elongation factor b (P-TEFb) complex, which results in reduction of serine 2 phosphorylation (Ser2P) in the RNA Pol II C-terminal domain and accumulation of RNA Pol II in the promoter-proximal region. Taken together, we uncover that m6A modifications of a non-coding RNA regulate RNA Pol II transcription and NSCLC tumorigenesis.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Factor B de Elongación Transcripcional Positiva/genética , Neoplasias Pulmonares/genética , ARN Nuclear Pequeño/genética , Transcripción Genética , Células HeLa , Metiltransferasas/genética , Metiltransferasas/metabolismoRESUMEN
Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA-target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3' end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.
Asunto(s)
MicroARNs , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Emparejamiento Base , MicroARNs/genética , MicroARNs/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/genéticaRESUMEN
There is a close regulatory relationship between the circadian clock and the abscisic acid (ABA) signaling pathway in regulating many developmental processes and stress responses. However, the exact feedback regulation mechanism between them is still poorly understood. Here, we identified the rice (Oryza sativa) clock component PSEUDO-RESPONSE REGULATOR 95 (OsPRR95) as a transcriptional regulator that accelerates seed germination and seedling growth by inhibiting ABA signaling. We also found that OsPRR95 binds to the ABA receptor gene REGULATORY COMPONENTS OF ABA RECEPTORS10 (OsRCAR10) DNA and inhibits its expression. Genetic analysis showed OsRCAR10 acts downstream of OsPRR95 in mediating ABA responses. In addition, the induction of OsPRR95 by ABA partly required a functional OsRCAR10, and the ABA-responsive element-binding factor ABSCISIC ACID INSENSITIVE5 (OsABI5) bound directly to the promoter of OsPRR95 and activated its expression, thus establishing a regulatory feedback loop between OsPRR95, OsRCAR10, and OsABI5. Taken together, our results demonstrated that the OsRCAR10-OsABI5-OsPRR95 feedback loop modulates ABA signaling to fine-tune seed germination and seedling growth, thus establishing the molecular link between ABA signaling and the circadian clock.
Asunto(s)
Arabidopsis , Relojes Circadianos , Oryza , Ácido Abscísico/metabolismo , Oryza/metabolismo , Relojes Circadianos/genética , Arabidopsis/genética , Germinación/fisiología , Plantones/metabolismo , Semillas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.
Asunto(s)
Factor de Transcripción Activador 4/genética , Neoplasias Colorrectales/genética , MicroARNs/genética , Ribonucleasa III/genética , Antagomirs/genética , Proteínas Argonautas/genética , Calnexina/genética , Movimiento Celular/genética , Proliferación Celular/genética , Neoplasias Colorrectales/patología , ARN Helicasas DEAD-box/genética , Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/genética , Factor 2 Eucariótico de Iniciación/genética , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Transducción de Señal/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Heading date is a key agronomic trait affecting crop yield. In rice, Early heading date 1 (Ehd1) is an important B-type response regulator in determination of heading date. Although many regulatory factors of Ehd1 expression have been functionally characterized, the direct regulators of Ehd1 largely remain to be identified. Here, we identified a new regulator of Ehd1, OsRE1, that directly binds to the A-box motif in the Ehd1 promoter. Osre1 confers an early heading phenotype due to elevated expression levels of Ehd1. OsRE1 is a nucleus-localized bZIP transcription factor with a diurnal rhythmic expression pattern. Furthermore, we identified an OsRE1-interacting protein, OsRIP1, and demonstrated that OsRIP1 can repress the transcript expression of Ehd1 in an OsRE1-dependent manner. Our genetic data showed that OsRE1 and OsRIP1 may function upstream of Ehd1 in regulating heading date. Together, our results suggest that OsRE1 functions cooperatively with OsRIP1 to regulate heading date through finely modulating the expression of Ehd1. In addition, OsRE1 and OsRIP1 are two minor heading date regulators, which are more desirable for fine-tuning heading date to improve rice regional adaptability.
Asunto(s)
Oryza , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Oryza/metabolismo , Fenotipo , Fotoperiodo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of â¼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.
Asunto(s)
Northern Blotting/métodos , Colorantes Fluorescentes/química , Hibridación de Ácido Nucleico/métodos , ARN/aislamiento & purificación , Sondas de ADN/química , ARN/química , Sondas ARN/químicaRESUMEN
MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) long and play important roles in post-transcriptional regulation in both plants and animals. In animals, precursor (pre-) miRNAs are â¼70 nt hairpins produced by Drosha cleavage of long primary (pri-) miRNAs in the nucleus. Exportin-5 (XPO5) transports pre-miRNAs into the cytoplasm for Dicer processing. Alternatively, pre-miRNAs containing a 5' 7-methylguanine (m7G-) cap can be generated independently of Drosha and XPO5. Here we identify a class of m7G-capped pre-miRNAs with 5' extensions up to 39 nt long. The 5'-extended pre-miRNAs are transported by Exportin-1 (XPO1). Unexpectedly, a long 5' extension does not block Dicer processing. Rather, Dicer directly cleaves 5'-extended pre-miRNAs by recognizing its 3' end to produce mature 3p miRNA and extended 5p miRNA both in vivo and in vitro. The recognition of 5'-extended pre-miRNAs by the Dicer Platform-PAZ-Connector (PPC) domain can be traced back to ancestral animal Dicers, suggesting that this previously unrecognized Dicer reaction mode is evolutionarily conserved. Our work reveals additional genetic sources for small regulatory RNAs and substantiates Dicer's essential role in RNAi-based gene regulation.
Asunto(s)
MicroARNs/metabolismo , Precursores del ARN/metabolismo , Ribonucleasa III/metabolismo , Sitio de Iniciación de la Transcripción , Animales , Línea Celular , Guanina/análogos & derivados , Carioferinas/metabolismo , Ratones , MicroARNs/química , Dominios Proteicos , Caperuzas de ARN , División del ARN , Interferencia de ARN , ARN Polimerasa II/metabolismo , Precursores del ARN/química , Procesamiento Postranscripcional del ARN , Transporte de ARN , ARN Bicatenario/química , ARN Bicatenario/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Ribonucleasa III/química , Proteína Exportina 1RESUMEN
Heading date is an important agronomic trait affecting crop yield. The GRAS protein family is a plant-specific super family extensively involved in plant growth and signal transduction. However, GRAS proteins are rarely reported have a role in regulating rice heading date. Here, we report a GRAS protein DHD1 (Delayed Heading Date1) delays heading and enhances yield in rice. Biochemical assays showed DHD1 physically interacts with OsHAP5C/D both in vitro and in vivo. DHD1 and OsHAP5C/D located in the nucleus and showed that rhythmic expression. Both DHD1 and OsHAP5C/D affect heading date by regulating expression of Ehd1. We propose that DHD1 interacts with OsHAP5C/D to delay heading date by inhibiting expression of Ehd1.
Asunto(s)
Flores/genética , Regulación de la Expresión Génica de las Plantas/genética , Oryza/genética , Proteínas de Plantas/metabolismo , Sitios de Carácter Cuantitativo/genética , Núcleo Celular/metabolismo , Regulación hacia Abajo , Flores/crecimiento & desarrollo , Flores/fisiología , Oryza/crecimiento & desarrollo , Oryza/fisiología , Fenotipo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Factores de Tiempo , Técnicas del Sistema de Dos HíbridosRESUMEN
The shoot apical meristem (SAM) consists of a population of multipotent cells that generates all aerial structures and regenerates itself. SAM maintenance and lateral organ development are regulated by several complex signaling pathways, in which the Argonaute gene-mediated pathway plays a key role. One Argonaute gene, AGO10, functions as a microRNA locker that attenuates miR165/166 activity and positively regulates shoot apical meristem development, but little is known about when and how AGO10 is regulated at the transcriptional level. In this work, we showed that transgenic rice plants overexpressing LBD12-1, an LBD family transcription factor, exhibited stunted growth, twisted leaves, abnormal anthers, and reduced SAM size. Further research revealed that LBD12-1 directly binds to the promoter region and represses the expression of AGO10. Overexpression of AGO10 in an LBD12-1 overexpression background rescued the growth defect phenotype of LBD12-1-overexpressing plants. The expression of LBD12-1 and its binding ability to the AGO10 promoter is induced by stress. lbd12-1 loss-of-function mutants showed similar phenotypes and SAM size to the wild type under normal conditions, but lbd12-1 had a larger SAM under salt stress. Our findings provide novel insights into the regulatory mechanism of AGO10 by which SAM size is controlled under stress conditions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Argonautas/genética , Meristema/crecimiento & desarrollo , Proteínas Represoras/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Argonautas/metabolismo , Regulación de la Expresión Génica de las Plantas , Meristema/genética , Filogenia , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Estrés Fisiológico/genética , Nicotiana/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
As a fundamental and dynamic cytoskeleton network, microfilaments (MFs) are regulated by diverse actin binding proteins (ABPs). Villins are one type of ABPs belonging to the villin/gelsolin superfamily, and their function is poorly understood in monocotyledonous plants. Here, we report the isolation and characterization of a rice (Oryza sativa) mutant defective in VILLIN2 (VLN2), which exhibits malformed organs, including twisted roots and shoots at the seedling stage. Cellular examination revealed that the twisted phenotype of the vln2 mutant is mainly caused by asymmetrical expansion of cells on the opposite sides of an organ. VLN2 is preferentially expressed in growing tissues, consistent with a role in regulating cell expansion in developing organs. Biochemically, VLN2 exhibits conserved actin filament bundling, severing and capping activities in vitro, with bundling and stabilizing activity being confirmed in vivo. In line with these findings, the vln2 mutant plants exhibit a more dynamic actin cytoskeleton network than the wild type. We show that vln2 mutant plants exhibit a hypersensitive gravitropic response, faster recycling of PIN2 (an auxin efflux carrier), and altered auxin distribution. Together, our results demonstrate that VLN2 plays an important role in regulating plant architecture by modulating MF dynamics, recycling of PIN2, and polar auxin transport.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Microfilamentos/metabolismo , Oryza/genética , Actinas/metabolismo , Transporte Biológico , Citoesqueleto/metabolismo , Genes Reporteros , Gravitropismo , Proteínas de Microfilamentos/genética , Mutación , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Oryza/ultraestructura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/ultraestructura , Plantas Modificadas Genéticamente , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Plantones/ultraestructuraRESUMEN
Heading date is an important agronomic trait related to crop yield. Many genes related to heading date have already been identified in rice (Oryza sativa), and a complicated, preliminary regulatory genetic network has also already been established, but the protein regulatory network is poorly understood. We have identified a novel heading date regulator, Heme Activator Protein like 1 (OsHAPL1), which inhibits flowering under long-day conditions. OsHAPL1 is a nuclear-localized protein that is highly expressed in leaves in a rhythmic manner. OsHAPL1 can physically interact with Days To Heading on chromosome 8 (DTH8), which physically interacts with Heading date 1 (Hd1) both in vitro and in vivo. OsHAPL1 forms a complex with DTH8 and Hd1 in Escherichia coli. OsHAPL1, DTH8, and Hd1 physically interact with the HAP complex, and also with general transcription factors in yeast (Saccharomyces cerevisiae). Further studies showed that OsHAPL1 represses the expression of the florigen genes and FLOWERING LOCUS T 1 (RFT1) and Hd3a through Early heading date 1 (Ehd1). We propose that OsHAPL1 functions as a transcriptional regulator and, together with DTH8, Hd1, the HAP complex, and general transcription factors, regulates the expression of target genes and then affects heading date by influencing the expression of Hd3a and RFT1 through Ehd1.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Oryza/genética , Proteínas de Plantas/genética , Secuencia de Bases , Flores/genética , Flores/crecimiento & desarrollo , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Success of modern agriculture relies heavily on breeding of crops with maximal regional adaptability and yield potentials. A major limiting factor for crop cultivation is their flowering time, which is strongly regulated by day length (photoperiod) and temperature. Here we report identification and characterization of Days to heading 7 (DTH7), a major genetic locus underlying photoperiod sensitivity and grain yield in rice. Map-based cloning reveals that DTH7 encodes a pseudo-response regulator protein and its expression is regulated by photoperiod. We show that in long days DTH7 acts downstream of the photoreceptor phytochrome B to repress the expression of Ehd1, an up-regulator of the "florigen" genes (Hd3a and RFT1), leading to delayed flowering. Further, we find that haplotype combinations of DTH7 with Grain number, plant height, and heading date 7 (Ghd7) and DTH8 correlate well with the heading date and grain yield of rice under different photoperiod conditions. Our data provide not only a macroscopic view of the genetic control of photoperiod sensitivity in rice but also a foundation for breeding of rice cultivars better adapted to the target environments using rational design.
Asunto(s)
Genes de Plantas , Oryza/genética , Fotoperiodo , Proteínas de Plantas/fisiología , Sitios de Carácter Cuantitativo , Adaptación Fisiológica , Agrobacterium tumefaciens/genética , Biomasa , Clonación Molecular , Cruzamientos Genéticos , Regulación de la Expresión Génica de las Plantas , Vectores Genéticos/genética , Haplotipos/genética , Oryza/crecimiento & desarrollo , Oryza/efectos de la radiación , Reguladores del Crecimiento de las Plantas/genética , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de TiempoRESUMEN
Flowering time determines the adaptability of crop plants to different local environments, thus being one of the most important agronomic traits targeted in breeding programs. Photoperiod is one of the key factors that control flowering in plant. A number of genes that participate in the photoperiod pathway have been characterized in long-day plants such as Arabidopsis, as well as in short-day plants such as Oryza sativa. Of those, CONSTANS (CO) as a floral integrator promotes flowering in Arabidopsis under long day conditions. In rice, Heading date1 (Hd1), a homologue of CO, functions in an opposite way, which inhibits flowering under long day conditions and induces flowering under short day conditions. Here, we show that another CONSTANS-like (COL) gene, OsCOL13, negatively regulates flowering in rice under both long and short day conditions. Overexpression of OsCOL13 delays flowering regardless of day length. We also demonstrated that OsCOL13 has a constitutive and rhythmic expression pattern, and that OsCOL13 is localized to the nucleus. OsCOL13 displays transcriptional activation activity in the yeast assays and likely forms homodimers in vivo. OsCOL13 suppresses the florigen genes Hd3a and RFT1 by repressing Ehd1, but has no relationship with other known Ehd1 regulators as determined by using mutants or near isogenic lines. In addition, the transcriptional level of OsCOL13 significantly decreased in the osphyb mutant, but remained unchanged in the osphya and osphyc mutants. Thus, we conclude that OsCOL13 functions as a negative regulator downstream of OsphyB and upstream of Ehd1 in the photoperiodic flowering in rice.
Asunto(s)
Oryza/metabolismo , Proteínas de Plantas/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Flores/genética , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Mutación , Oryza/genética , Proteínas de Plantas/genéticaRESUMEN
Flowering time, or heading date, is a critical agronomic trait that determines the cropping season and regional adaptability, and ultimately grain yield in rice. A number of genes involved in photoperiodic flowering have been cloned and their roles in modulating expression of the flowering genes have been characterized to a certain extent. However, much less is known about the pathway in transmitting the day length response signal(s) to induce transition to reproductive growth. Here, we report a constitutive flowering repressor OsCOL10, which encodes a member of the CONSTANS-like (COL) family. Transgenic rice plants overexpressing OsCOL10 (driven by a strong promoter or by fusing it to the activation domain of VP64) showed delayed flowering time under both short and long days.OsCOL10 is affected by the circadian clock and is preferentially expressed in leaf mesophyll cells; it is localized to the nucleus and has transcriptional activation activity. Further studies show that OsCOL10 represses the expression of theFT-like genes RFT1 and Hd3a through Ehd1. Transcripts of OsCOL10 are more abundant in plants carrying a functional Ghd7 allele or overexpressing Ghd7 than in Ghd7-deficient plants, thus placing OsCOL10 downstream of Ghd7.Taking these findings together, we conclude that OsCOL10 functions as a flowering time repressor that links Ghd7 and Ehd1 in rice.
Asunto(s)
Flores/genética , Regulación de la Expresión Génica de las Plantas , Oryza/fisiología , Proteínas de Plantas/genética , Núcleo Celular/metabolismo , Relojes Circadianos/genética , Células del Mesófilo/fisiología , Oryza/genética , Fotoperiodo , Hojas de la Planta/citología , Hojas de la Planta/fisiología , Proteínas de Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Meiosis is essential for gametogenesis in sexual reproduction in rice (Oryza sativa L.). We identified a MutS-homolog (MSH) family gene OsMSH4 in a trisomic plant. Cytological analysis showed that developments of both pollen and embryo sacs in an Osmsh4 mutant were blocked due to defective chromosome pairing. Compared with the wild type, the Osmsh4 mutant displayed a significant ~21.9% reduction in chiasma frequency, which followed a Poisson distribution, suggesting that class I crossover formation in the mutant was impaired. Temporal and spatial expression pattern analyses showed that OsMSH4 was preferentially expressed in meiocytes during their meiosis, indicating a critical role in gametogenesis. Subcellular localization showed that OsMSH4-green fluorescent protein was predominantly located in the nucleus. OsMSH4 could interact with another MSH member (OsMSH5) through the N-terminus and C-terminus, respectively. Direct physical interaction between OsMSH5, OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c was identified by yeast two-hybrid assays and further validated by pull-down assays. Our results supported the conclusion that the OsMSH4/5 heterodimer plays a key role in regulation of crossover formation during rice meiosis by interaction with the RPA complex.
Asunto(s)
Gametogénesis en la Planta , Meiosis , Oryza/citología , Oryza/metabolismo , Óvulo Vegetal/metabolismo , Proteínas de Plantas/metabolismo , Polen/metabolismo , Emparejamiento Cromosómico , Cromosomas de las Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Prueba de Complementación Genética , Mutación/genética , Oryza/embriología , Oryza/genética , Óvulo Vegetal/ultraestructura , Proteínas de Plantas/genética , Polen/ultraestructura , Unión Proteica , Multimerización de Proteína , Transporte de Proteínas , Fracciones Subcelulares/metabolismoRESUMEN
Land plants have evolved increasingly complex regulatory modes of their flowering time (or heading date in crops). Rice (Oryza sativa L.) is a short-day plant that flowers more rapidly in short-day but delays under long-day conditions. Previous studies have shown that the CO-FT module initially identified in long-day plants (Arabidopsis) is evolutionary conserved in short-day plants (Hd1-Hd3a in rice). However, in rice, there is a unique Ehd1-dependent flowering pathway that is Hd1-independent. Here, we report isolation and characterization of a positive regulator of Ehd1, Early heading date 4 (Ehd4). ehd4 mutants showed a never flowering phenotype under natural long-day conditions. Map-based cloning revealed that Ehd4 encodes a novel CCCH-type zinc finger protein, which is localized to the nucleus and is able to bind to nucleic acids in vitro and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional regulator. Ehd4 expression is most active in young leaves with a diurnal expression pattern similar to that of Ehd1 under both short-day and long-day conditions. We show that Ehd4 up-regulates the expression of the "florigen" genes Hd3a and RFT1 through Ehd1, but it acts independently of other known Ehd1 regulators. Strikingly, Ehd4 is highly conserved in the Oryza genus including wild and cultivated rice, but has no homologs in other species, suggesting that Ehd4 is originated along with the diversification of the Oryza genus from the grass family during evolution. We conclude that Ehd4 is a novel Oryza-genus-specific regulator of Ehd1, and it plays an essential role in photoperiodic control of flowering time in rice.
Asunto(s)
Flores , Proteínas Nucleares/genética , Oryza , Fotoperiodo , Proteínas de Plantas , Transactivadores/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Oryza/crecimiento & desarrollo , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Drought is a recurring climatic hazard that reduces the crop yields. To avoid the negative effects of drought on crop production, extensive efforts have been devoted to investigating the complex mechanisms of gene expression and signal transduction during drought stress. Receptor-like kinases (RLKs) play important roles in perceiving extracellular stimuli and activating downstream signalling responses. The rice genome contains >1100 RLK genes, of which only two are reported to function in drought stress. A leucine-rich repeat (LRR)-RLK gene named Leaf Panicle 2 (LP2) was previously found to be strongly expressed in leaves and other photosynthetic tissues, but its function remains unclear. In the present study, it was shown that the expression of LP2 was down-regulated by drought and abscisic acid (ABA). Transgenic plants overexpressing LP2 accumulated less H2O2, had more open stomata in leaves, and showed hypersensitivity to drought stress. Further investigation revealed that transcription of LP2 was directly regulated by the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST). In addition, LP2 was identified as a functional kinase localized to the plasma membrane and interacted with the drought-responsive aquaporin proteins OsPIP1; 1, OsPIP1; 3, and OsPIP2; 3. Thus, the findings provided evidence that the LRR-RLK LP2, transcriptionally regulated by the drought-related transcription factor DST, served as a negative regulator in drought response.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Oryza/crecimiento & desarrollo , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/genética , Ácido Abscísico/metabolismo , Adaptación Biológica , Sequías , Oryza/metabolismo , Filogenia , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Estructura Terciaria de Proteína , Factores de Transcripción/metabolismoRESUMEN
KEY MESSAGE: Mutation of the AM1 gene causes an albino midrib phenotype and enhances tolerance to drought in rice K(+) efflux antiporter (KEA) genes encode putative potassium efflux antiporters that are mainly located in plastid-containing organisms, ranging from lower green algae to higher flowering plants. However, little genetic evidence has been provided on the functions of KEA in chloroplast development. In this study, we isolated a rice mutant, albino midrib 1 (am1), with green- and white-variegation in the first few leaves, and albino midrib phenotype in older tissues. We found that AM1 encoded a putative KEA in chloroplast. AM1 was highly expressed in leaves, while lowly in roots. Chloroplast gene expression and proteins accumulation were affected during chlorophyll biosynthesis and photosynthesis in am1 mutants. Interestingly, AM1 was induced by salt and PEG, and am1 showed enhanced sensitivity to salinity in seed germination and increased tolerance to drought. Taken together, we concluded that KEAs were involved in chloroplast development and played important roles in drought tolerance.
Asunto(s)
Antiportadores/genética , Cloroplastos/fisiología , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Potasio/metabolismo , Secuencia de Aminoácidos , Antiportadores/metabolismo , Clorofila/metabolismo , Sequías , Germinación , Datos de Secuencia Molecular , Mutación , Oryza/fisiología , Oryza/ultraestructura , Fenotipo , Fotosíntesis , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Hojas de la Planta/ultraestructura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión , Salinidad , Plantones/genética , Plantones/fisiología , Plantones/ultraestructura , Semillas/genética , Semillas/fisiología , Semillas/ultraestructura , Alineación de SecuenciaRESUMEN
RNA interference (RNAi) is a powerful tool for sequence-specific gene knockdown in therapeutic and research applications. However, spatiotemporal control of RNAi is required to decrease nonspecific targeting, potential toxicity, and allow targeting of essential genes. Herein we describe a class of de-novo-designed RNA switches that enable sequence-specific regulation of RNAi in mammalian cells. Using cis-repressing RNA elements, we engineer RNA devices that only initiate microRNA biogenesis when binding with cognate trigger RNAs. We demonstrate that this conditional RNAi system, termed Orthogonal RNA Interference induced by Trigger RNA (ORIENTR), provides up to 14-fold increases in artificial miRNA biogenesis upon activation in orthogonal libraries. We show that integration of ORIENTR triggers with dCas13d enhances dynamic range to up to 31-fold. We further demonstrate that ORIENTR can be applied to detect endogenous RNA signals and to conditionally knockdown endogenous genes, thus enabling regulatory possibilities including cell-type-specific RNAi and rewiring of transcriptional networks via RNA profile.
Asunto(s)
MicroARNs , Interferencia de ARN , Activación Transcripcional , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Células HEK293 , Animales , Técnicas de Silenciamiento del Gen , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN/metabolismo , ARN/genéticaRESUMEN
MicroRNAs (miRNA) load onto AGO proteins to target mRNAs for translational repression or degradation. However, miRNA degradation can be triggered when extensively base-paired with target RNAs, which induces confirmational change of AGO and recruitment of ZSWIM8 ubiquitin ligase to mark AGO for proteasomal degradation. This target RNA-directed miRNA degradation (TDMD) mechanism appears to be evolutionarily conserved, but recent studies have focused on mammalian systems. Here, we performed AGO1-CLASH in Drosophila S2 cells, with Dora (ortholog of vertebrate ZSWIM8) knockout mediated by CRISPR-Cas9 to identify five TDMD triggers (sequences that can induce miRNA degradation). Interestingly, one trigger in the 3' UTR of AGO1 mRNA induces miR-999 degradation. CRISPR-Cas9 knockout of the AGO1 trigger in S2 cells and in Drosophila specifically elevates miR-999, with concurrent repression of the miR-999 targets. AGO1 trigger knockout flies respond poorly to hydrogen peroxide-induced stress, demonstrating the physiological importance of this TDMD event.