RESUMO
MicroRNAs (miRNAs) are essential regulators of gene expression, defined by their unique biogenesis, which requires the precise excision of the small RNA from an imperfect fold-back precursor. Unlike their animal counterparts, plant miRNA precursors exhibit variations in sizes and shapes. Plant MIRNAs can undergo processing in a base-to-loop or loop-to-base direction, with DICER-LIKE1 (DCL1) releasing the miRNA after two cuts (two-step MIRNAs) or more (sequential MIRNAs). In this study, we demonstrate the critical role of the miRNA/miRNA* duplex region in the processing of miRNA precursors. We observed that endogenous MIRNAs frequently experience suboptimal processing in vivo due to mismatches in the miRNA/miRNA* duplex, a key region that fine-tunes miRNA levels. Enhancing the interaction energy of the miRNA/miRNA* duplex in two-step MIRNAs results in a substantial increase in miRNA levels. Conversely, sequential MIRNAs display distinct and specific requirements for the miRNA/miRNA* duplexes along their foldback structure. Our work establishes a connection between the miRNA/miRNA* structure and precursor processing mechanisms. Furthermore, we reveal a link between the biological function of miRNAs and the processing mechanism of their precursors with the evolution of plant miRNA/miRNA* duplex structures.
Assuntos
MicroRNAs , Processamento Pós-Transcricional do RNA , RNA de Plantas , Ribonuclease III , MicroRNAs/genética , MicroRNAs/metabolismo , RNA de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/química , Ribonuclease III/metabolismo , Ribonuclease III/genética , Precursores de RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/química , Regulação da Expressão Gênica de Plantas , Arabidopsis/genética , Arabidopsis/metabolismo , Conformação de Ácido Nucleico , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo CelularRESUMO
The growth-regulating factor (GRF) family of transcriptional factors are involved in the control of leaf size and senescence, inflorescence and root growth, grain size, and plant regeneration. However, there is limited information about the genes regulated by these transcriptional factors, which are in turn responsible for their functions. Using a meta-analysis approach, we identified genes encoding Arabidopsis (Arabidopsis thaliana) zinc-finger homeodomain (ZF-HD) transcriptional factors, as potential targets of the GRFs. We further showed that GRF3 binds to the promoter of one of the members of the ZF-HD family, HOMEOBOX PROTEIN 33 (HB33), and activates its transcription. Increased levels of HB33 led to different modifications in leaf cell number and size that were dependent on its expression levels. Furthermore, we found that expression of HB33 for an extended period during leaf development increased leaf longevity. To cope with the functional redundancy among ZF-HD family members, we generated a dominant repressor version of HB33, HB33-SRDX. Expression of HB33-SRDX from HB33 regulatory regions was seedling-lethal, revealing the importance of the ZF-HD family in plant development. Misexpression of HB33-SRDX in early leaf development caused a reduction in both cell size and number. Interestingly, the loss-of-function of HB33 in lines carrying a GRF3 allele insensitive to miR396 reverted the delay in leaf senescence characteristic of these plants. Our results revealed functions for ZF-HDs in leaf development and linked them to the GRF pathway.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , MicroRNAs , Arabidopsis/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , MicroRNAs/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Folhas de Planta/metabolismoRESUMO
Plants rely on complex regulatory mechanisms to ensure proper growth and development. As plants are sessile organisms, these mechanisms must be flexible enough to adapt to changes in the environment. GROWTH-REGULATING FACTORS (GRFs) are plant-specific transcription factors that act as a central hub controlling plant growth and development, which offer promising biotechnological applications to enhance plant performance. Here, we analyze the complex molecular mechanisms that regulate GRFs activity, and how their natural and synthetic variants can impact on plant growth and development. We describe the biological roles of the GRFs and examine how they regulate gene expression and contribute to the control of organ growth and plant responses to a changing environment. This review focuses on the premise that unlocking the full biotechnological potential of GRFs requires a thorough understanding of the various regulatory layers governing GRF activity, the functional divergence among GRF family members, and the gene networks that they regulate.
Assuntos
Regulação da Expressão Gênica de Plantas , Desenvolvimento Vegetal , Desenvolvimento Vegetal/genética , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Meio Ambiente , Plantas/metabolismo , Plantas/genéticaRESUMO
Gene expression is reconfigured rapidly during the cell cycle to execute the cellular functions specific to each phase. Studies conducted with synchronized plant cell suspension cultures have identified hundreds of genes with periodic expression patterns across the phases of the cell cycle, but these results may differ from expression occurring in the context of intact organs. Here, we describe the use of fluorescence-activated cell sorting to analyze the gene expression profile of G2/M cells in the growing root. To this end, we isolated cells expressing the early mitosis cell cycle marker CYCLINB1;1-GFP from Arabidopsis root tips. Transcriptome analysis of these cells allowed identification of hundreds of genes whose expression is reduced or enriched in G2/M cells, including many not previously reported from cell suspension cultures. From this dataset, we identified SCL28, a transcription factor belonging to the GRAS family, whose messenger RNA accumulates to the highest levels in G2/M and is regulated by MYB3R transcription factors. Functional analysis indicates that SCL28 promotes progression through G2/M and modulates the selection of cell division planes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Mitose , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Meristema/metabolismo , Mitose/genética , Organogênese , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
KEY MESSAGE: Proper root growth depends on the clearance of TCP transcripts from the root apical meristem by microRNA miR319. The evolutionarily conserved microRNA miR319 regulates genes encoding TCP transcription factors in angiosperms. The miR319-TCP module controls cell proliferation and differentiation in leaves and other aerial organs. The current model sustains that miR319 quantitatively tunes TCP activity during leaf growth and development, ultimately affecting its size. In this work we studied how this module participates in Arabidopsis root development. We found that misregulation of TCP activity through impairment of miR319 binding decreased root meristem size and root length. Cellular and molecular analyses revealed that high TCP activity affects cell number and cyclin expression but not mature cell length, indicating that, in roots, unchecking the expression of miR319-regulated TCPs significantly affects cell proliferation. Conversely, tcp multiple mutants showed no obvious effect on root growth, but strong defects in leaf morphogenesis. Therefore, in contrast to the quantitative regulation of the TCPs by miR319 in leaves, our data suggest that miR319 clears TCP transcripts from root cells. Hence, we provide new insights into the functions of the miR319-TCP regulatory system in Arabidopsis development, highlighting a different modus operandi for its action mechanism in roots and shoots.
Assuntos
Proteínas de Arabidopsis/fisiologia , MicroRNAs/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , MicroRNAs/metabolismo , Microscopia Confocal , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Members of the GROWTH REGULATING FACTOR (GRF) family of transcription factors play key roles in the promotion of plant growth and development. Many GRFs are post-transcriptionally repressed by microRNA (miRNA) miR396, an evolutionarily conserved small RNA, which restricts their expression to proliferative tissue. We performed a comprehensive analysis of the GRF family in eudicot plants and found that in many species all the GRFs have a miR396-binding site. Yet, we also identified GRFs with mutations in the sequence recognized by miR396, suggesting a partial or complete release of their post-transcriptional repression. Interestingly, Brassicaceae species share a group of GRFs that lack miR396 regulation, including Arabidopsis GRF5 and GRF6. We show that instead of miR396-mediated post-transcriptional regulation, the spatiotemporal control of GRF5 is achieved through evolutionarily conserved promoter sequences, and that AUXIN RESPONSE FACTOR 2 (ARF2) binds to such conserved sequences to repress GRF5 expression. Furthermore, we demonstrate that the unchecked expression of GRF5 in arf2 mutants is responsible for the increased cell number of arf2 leaves. The results describe a switch in the repression mechanisms that control the expression of GRFs and mechanistically link the control of leaf growth by miR396, GRFs, and ARF2 transcription factors.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Magnoliopsida/crescimento & desenvolvimento , Magnoliopsida/genética , MicroRNAs , Reguladores de Crescimento de Plantas/genética , Fatores de Transcrição/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Genótipo , Desenvolvimento Vegetal , Análise de Sequência de ProteínaRESUMO
Seeds germinating underground display a specific developmental programme, termed skotomorphogenesis, to ensure survival of the emerging seedlings until they reach the light. They rapidly elongate the hypocotyl and maintain the cotyledons closed, forming a hook with the hypocotyl in order to protect apical meristematic cells from mechanical damage. Such crucial events for the fate of the seedling are tightly regulated and although some transcriptional regulators and phytohormones are known to be implicated in this regulation, we are still far from a complete understanding of these biological processes. Our work provides information on the diverse roles in skotomorphogenesis of the core components of microRNA biogenesis in Arabidopsis, HYL1, DCL1, and SE. We show that hypocotyl elongation is promoted by all these components, probably through the action of specific miRNAs. Hook development also depends on these proteins however, remarkably, HYL1 exerts its role in an opposite way to DCL1 and SE. Interestingly, we found that a specific HYL1 domain involved in protein-protein interaction is required for this function. Genetic evidences also point to the phosphorylation status of HYL1 as important for this function. We propose that HYL1 help maintain the hook closed during early skotomorphogenesis in a microprocessor-independent manner by repressing the activity of HY5, the transcriptional master regulator that triggers light responses. This work uncovers a previously unnoticed link between components of the miRNA biogenesis machinery, the skotomorphogenic growth, and hook development in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
Agrobacterium T-DNA-encoded 6B proteins cause remarkable growth effects in plants. Nicotiana otophora carries two cellular T-DNAs with three slightly divergent 6b genes (TE-1-6b-L, TE-1-6b-R and TE-2-6b) originating from a natural transformation event. In Arabidopsis thaliana, expression of 2×35S:TE-2-6b, but not 2×35S:TE-1-6b-L or 2×35S:TE-1-6b-R, led to plants with crinkly leaves, which strongly resembled mutants of the miR319a/TCP module. This module is composed of MIR319A and five CIN-like TCP (TEOSINTHE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR) genes (TCP2, TCP3, TCP4, TCP10 and TCP24) targeted by miR319a. The CIN-like TCP genes encode transcription factors and are required for cell division arrest at leaf margins during development. MIR319A overexpression causes excessive growth and crinkly leaves. TE-2-6b plants did not show increased miR319a levels, but the mRNA levels of the TCP4 target gene LOX2 were decreased, as in jaw-D plants. Co-expression of green fluorescent protein (GFP)-tagged TCPs with native or red fluorescent protein (RFP)-tagged TE-6B proteins led to an increase in TCP protein levels and formation of numerous cytoplasmic dots containing 6B and TCP proteins. Yeast double-hybrid experiments confirmed 6B/TCP binding and showed that TE-1-6B-L and TE-1-6B-R bind a smaller set of TCP proteins than TE-2-6B. A single nucleotide mutation in TE-1-6B-R enlarged its TCP-binding repertoire to that of TE-2-6B and caused a crinkly phenotype in Arabidopsis. Deletion analysis showed that TE-2-6B targets the TCP4 DNA-binding domain and directly interferes with transcriptional activation. Taken together, these results provide detailed insights into the mechanism of action of the N. otophora TE-encoded 6b genes.
Assuntos
Agrobacterium/metabolismo , Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Arabidopsis/microbiologia , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/metabolismo , Perfilação da Expressão Gênica , Microscopia Confocal , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Reação em Cadeia da Polimerase , Nicotiana/metabolismo , Nicotiana/microbiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
In the root meristem, the quiescent center (QC) is surrounded by stem cells, which in turn generate the different cell types of the root. QC cells rarely divide under normal conditions but can replenish damaged stem cells. In the proximal meristem, the daughters of stem cells, which are referred to as transit-amplifying cells, undergo additional rounds of cell division prior to differentiation. Here, we describe the functions of GRF-INTERACTING FACTORs (GIFs), including ANGUSTIFOLIA3 (AN3), in Arabidopsis thaliana roots. GIFs have been shown to interact with GRF transcription factors and SWI/SNF chromatin remodeling complexes. We found that combinations of GIF mutants cause the loss of QC identity. However, despite their QC impairment, GIF mutants have a significantly enlarged root meristem with additional lateral root cap layers. We show that the increased expression of PLETHORA1 (PLT1) is at least partially responsible for the large root meristems of an3 mutants. Furthermore, we found that GIFs are necessary for maintaining the precise expression patterns of key developmental regulators and that AN3 complexes bind directly to the promoter regions of PLT1 as well as SCARECROW We propose that AN3/GIFs participate in different pathways that control QC organization and the size of the meristem.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Diferenciação Celular/genética , Divisão Celular/genética , Montagem e Desmontagem da Cromatina/genética , Homeostase/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/fisiologia , Mutação , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression. Their biogenesis relies on the cleavage of longer precursors by a nuclear localized processing machinery. The evolutionary preference of plant miRNAs to silence transcription factors turned these small molecules into key actors during growth and adaptive responses. Furthermore, during their life cycle plants are subject to changes in the environmental conditions surrounding them. In order to face these changes, plants display unique adaptive capacities based on an enormous developmental plasticity, where miRNAs play central roles. Many individual miRNAs have been shown to modulate the plant response to different environmental cues and stresses. In the last few years, increasing evidence has shown that not only individual genes encoding miRNAs but also the miRNA pathway as a whole is subject to regulation in response to external stimulus. In this review, we discuss the current knowledge about the miRNA pathway. We dissect the pathway to analyze the events leading to the generation of these small RNAs and emphasize the regulation of core components of the miRNA biogenesis machinery.
Assuntos
MicroRNAs/biossíntese , Plantas/metabolismo , RNA de Plantas/biossíntese , Estresse Fisiológico/fisiologia , Regulação da Expressão Gênica de Plantas , Redes e Vias Metabólicas , MicroRNAs/genética , Proteínas de Plantas , Plantas/genética , Processamento Pós-Transcricional do RNA , RNA de Plantas/genética , Fatores de TranscriçãoRESUMO
MicroRNAs (miRNAs) are endogenous small RNAs that recognize target sequences by base complementarity and play a role in the regulation of target gene expression. They are processed from longer precursor molecules that harbor a fold-back structure. Plant miRNA precursors are quite variable in size and shape, and are recognized by the processing machinery in different ways. However, ancient miRNAs and their binding sites in target genes are conserved during evolution. Here, we designed a strategy to systematically analyze MIRNAs from different species generating a graphical representation of the conservation of the primary sequence and secondary structure. We found that plant MIRNAs have evolutionary footprints that go beyond the small RNA sequence itself, yet their location along the precursor depends on the specific MIRNA We show that these conserved regions correspond to structural determinants recognized during the biogenesis of plant miRNAs. Furthermore, we found that the members of the miR166 family have unusual conservation patterns and demonstrated that the recognition of these precursors in vivo differs from other known miRNAs. Our results describe a link between the evolutionary conservation of plant MIRNAs and the mechanisms underlying the biogenesis of these small RNAs and show that the MIRNA pattern of conservation can be used to infer the mode of miRNA biogenesis.
Assuntos
Evolução Molecular , MicroRNAs/genética , RNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Estabilidade de RNARESUMO
Many evolutionarily conserved microRNAs (miRNAs) in plants regulate transcription factors with key functions in development. Hence, mutations in the core components of the miRNA biogenesis machinery cause strong growth defects. An essential aspect of miRNA biogenesis is the precise excision of the small RNA from its precursor. In plants, miRNA precursors are largely variable in size and shape and can be processed by different modes. Here, we optimized an approach to detect processing intermediates during miRNA biogenesis. We characterized a miRNA whose processing is triggered by a terminal branched loop. Plant miRNA processing can be initiated by internal bubbles, small terminal loops or branched loops followed by dsRNA segments of 15-17 bp. Interestingly, precision and efficiency vary with the processing modes. Despite the various potential structural determinants present in a single a miRNA precursor, DCL1 is mostly guided by a predominant structural region in each precursor in wild-type plants. However, our studies in fiery1, hyl1 and se mutants revealed the existence of cleavage signatures consistent with the recognition of alternative processing determinants. The results provide a general view of the mechanisms underlying the specificity of miRNA biogenesis in plants.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , MicroRNAs/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas de Ligação a RNA/genética , Sítios de Ligação , Biologia Computacional , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , MicroRNAs/biossíntese , Mutação , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Processamento Pós-Transcricional do RNA , RNA de Cadeia Dupla/genética , Plântula , Transcrição Gênica , TransgenesRESUMO
The characteristic leaf shapes we see in all plants are in good part the outcome of the combined action of several transcription factor networks that translate into cell division activity during the early development of the organ. We show here that wild-type leaves have distinct transcriptomic profiles in center and marginal regions. Certain transcripts are enriched in margins, including those of CINCINNATA-like TCPs (TEOSINTE BRANCHED, CYCLOIDEA and PCF1/2) and members of the NGATHA and STYLISH gene families. We study in detail the contribution of microRNA319 (miR319)-regulated TCP transcription factors to the development of the center and marginal regions of Arabidopsis (Arabidopsis thaliana) leaves. We compare in molecular analyses the wild type, the tcp2 tcp4 mutant that has enlarged flat leaves, and the tcp2 tcp3 tcp4 tcp10 mutant with strongly crinkled leaves. The different leaf domains of the tcp mutants show changed expression patterns for many photosynthesis-related genes, indicating delayed differentiation, especially in the marginal parts of the organ. At the same time, we found an up-regulation of cyclin genes and other genes that are known to participate in cell division, specifically in the marginal regions of tcp2 tcp3 tcp4 tcp10 Using GUS reporter constructs, we confirmed extended mitotic activity in the tcp2 tcp3 tcp4 tcp10 leaf, which persisted in small defined foci in the margins when the mitotic activity had already ceased in wild-type leaves. Our results describe the role of miR319-regulated TCP transcription factors in the coordination of activities in different leaf domains during organ development.
Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
To ensure an adequate organ mass, the daughters of stem cells progress through a transit-amplifying phase displaying rapid cell division cycles before differentiating. Here, we show that Arabidopsis thaliana microRNA miR396 regulates the transition of root stem cells into transit-amplifying cells by interacting with GROWTH-REGULATING FACTORs (GRFs). The GRFs are expressed in transit-amplifying cells but are excluded from the stem cells through inhibition by miR396. Inactivation of the GRFs increases the meristem size and induces periclinal formative divisions in transit-amplifying cells. The GRFs repress PLETHORA (PLT) genes, regulating their spatial expression gradient. Conversely, PLT activates MIR396 in the stem cells to repress the GRFs. We identified a pathway regulated by GRF transcription factors that represses stem cell-promoting genes in actively proliferating cells, which is essential for the progression of the cell cycle and the orientation of the cell division plane. If unchecked, the expression of the GRFs in the stem cell niche suppresses formative cell divisions and distorts the organization of the quiescent center. We propose that the interactions identified here between miR396 and GRF and PLT transcription factors are necessary to establish the boundary between the stem cell niche and the transit-amplifying region.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Divisão Celular , Meristema/citologia , Meristema/genética , Meristema/crescimento & desenvolvimento , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Nicho de Células-Tronco/genética , Células-Tronco , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
MicroRNAs (miRNAs) derive from longer precursors with fold-back structures. While animal miRNA precursors have homogenous structures, plant precursors comprise a collection of fold-backs with variable size and shape. Here, we design an approach to systematically analyze miRNA processing intermediates and characterize the biogenesis of most of the evolutionarily conserved miRNAs present in Arabidopsis thaliana. We found that plant miRNAs are processed by four mechanisms, depending on the sequential direction of the processing machinery and the number of cuts required to release the miRNA. Classification of the precursors according to their processing mechanism revealed specific structural determinants for each group. We found that the complexity of the miRNA processing pathways occurs in both ancient and evolutionarily young sequences and that members of the same family can be processed in different ways. We observed that different structural determinants compete for the processing machinery and that alternative miRNAs can be generated from a single precursor. The results provide an explanation for the structural diversity of miRNA precursors in plants and new insights toward the understanding of the biogenesis of small RNAs.
Assuntos
Arabidopsis/metabolismo , MicroRNAs/química , MicroRNAs/metabolismo , RNA de Plantas/metabolismo , Arabidopsis/química , Arabidopsis/genética , Sequência de Bases , Sequência Conservada , Evolução Molecular , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Conformação de Ácido Nucleico , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Plantas/química , RNA de Plantas/genéticaRESUMO
Because of their sessile lifestyle, plants are continuously exposed to solar UV-B radiation. Inhibition of leaf growth is one of the most consistent responses of plants upon exposure to UV-B radiation. In this work, we investigated the role of Growth-Regulating Factors (GRFs) and of microRNA miR396 in UV-B-mediated inhibition of leaf growth in Arabidopsis thaliana plants. We demonstrate that miRNA396 is upregulated by UV-B radiation in proliferating tissues and that this induction is correlated with a decrease in GRF1, GRF2, and GRF3 transcripts. Induction of miR396 results in inhibition of cell proliferation, and this outcome is independent of the UV-B photoreceptor UV resistance locus 8, as well as ATM AND RAD3-related and the mitogen-activated protein kinase MPK6, but is dependent on MPK3. Transgenic plants expressing an artificial target mimic directed against miR396 (MIM396) with a decrease in the endogenous microRNA activity or plants expressing miR396-resistant copies of several GRFs are less sensitive to this inhibition. Consequently, at intensities that can induce DNA damage in Arabidopsis plants, UV-B radiation limits leaf growth by inhibiting cell division in proliferating tissues, a process mediated by miR396 and GRFs.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Divisão Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , MicroRNAs/metabolismo , Modelos Biológicos , Fotorreceptores de Plantas/genética , Fotorreceptores de Plantas/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Plantas Geneticamente Modificadas , Raios UltravioletaRESUMO
The growth-regulating factors (GRFs) are plant-specific transcription factors. They form complexes with GRF-interacting factors (GIFs), a small family of transcriptional co-activators. In Arabidopsis thaliana, seven out of the nine GRFs are controlled by microRNA miR396. Analysis of Arabidopsis plants carrying a GRF3 allele insensitive to miR396 revealed a strong boost in the number of cells in leaves, which was further enhanced synergistically by an additional increase of GIF1 levels. Genetic experiments revealed that GRF3 can still increase cell number in gif1 mutants, albeit to a much lesser extent. Genome-wide transcript profiling indicated that the simultaneous increase of GRF3 and GIF1 levels causes additional effects in gene expression compared to either of the transgenes alone. We observed that GIF1 interacts in vivo with GRF3, as well as with chromatin-remodeling complexes, providing a mechanistic explanation for the synergistic activities of a GRF3-GIF1 complex. Interestingly, we found that, in addition to the leaf size, the GRF system also affects the organ longevity. Genetic and molecular analysis revealed that the functions of GRFs in leaf growth and senescence can be uncoupled, demonstrating that the miR396-GRF-GIF network impinges on different stages of leaf development. Our results integrate the post-transcriptional control of the GRF transcription factors with the progression of leaf development.
Assuntos
Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/genética , Folhas de Planta/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis , Senescência Celular/genética , Senescência Celular/fisiologia , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
MOTIVATION: MicroRNAs (miRNAs) are major regulators of gene expression in plants and animals. They recognize their target messenger RNAs (mRNAs) by sequence complementarity and guide them to cleavage or translational arrest. So far, the prediction of plant miRNA-target pairs generally relies on the use of empirical parameters deduced from known miRNA-target interactions. RESULTS: We developed comTAR, a web tool for the prediction of miRNA targets that is mainly based on the conservation of the potential regulation in different species. We used data generated from a pipeline applied to transcript datasets of 33 angiosperms that was used to build a database of potential miRNA targets of different plant species. The database contains information describing each miRNA-target pair, their function and evolutionary conservation, while the results are displayed in a user-friendly interface. The tool also allows the search using new miRNAs. AVAILABILITY AND IMPLEMENTATION: The Web site is free to all users, with no login requirements, at http://rnabiology.ibr-conicet.gov.ar/comtar.
Assuntos
MicroRNAs/química , RNA Mensageiro/química , RNA de Plantas/química , Software , Animais , Sequência de Bases , Sequência Conservada , Internet , Magnoliopsida/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismoRESUMO
MicroRNAs (miRNAs) are small RNAs that derive from endogenous precursors harboring foldback structures. Plant miRNA precursors are quite variable in their size and shape. Still, the miRNA processing machinery, consisting of DICER-LIKE1 (DCL1) and accessory proteins recognize structural features on the precursors to cleave them at specific places releasing the mature miRNAs. The identification of miRNA processing intermediates in plants has mostly relied on a modified 5' RACE method, designed to detect the 5' end of uncapped RNAs. However, this method is time consuming and is, therefore, only practical for the analysis of a handful miRNAs. Here, we present a modification of this approach in order to perform genome-wide analysis of miRNA processing intermediates. Briefly, a reverse transcription is performed with a mixture of specific primers designed against all known miRNA precursors. miRNA processing intermediates are then specifically amplified to generate a library and subjected to deep sequencing. This method, called SPARE (Specific Parallel Amplification of 5' RNA Ends) allows the identification of processing intermediates for most of the Arabidopsis miRNAs. The results enable the determination of the DCL1 processing direction and the cleavage sites introduced by miRNA processing machinery in the precursors. The SPARE method can be easily adapted to detect miRNA-processing intermediates in other systems.
RESUMO
MicroRNAs (miRNAs) are â¼21 nt small RNAs that regulate gene expression in animals and plants. They can be grouped into families comprising different genes encoding similar or identical mature miRNAs. Several miRNA families are deeply conserved in plant lineages and regulate key aspects of plant development, hormone signaling, and stress response. The ancient miRNA miR396 regulates conserved targets belonging to the GROWTH-REGULATING FACTOR (GRF) family of transcription factors, which are known to control cell proliferation in Arabidopsis leaves. In this work, we characterized the regulation of an additional target for miR396, the transcription factor bHLH74, that is necessary for Arabidopsis normal development. bHLH74 homologs with a miR396 target site could only be detected in the sister families Brassicaceae and Cleomaceae. Still, bHLH74 repression by miR396 is required for margin and vein pattern formation of Arabidopsis leaves. MiR396 contributes to the spatio-temporal regulation of GRF and bHLH74 expression during leaf development. Furthermore, a survey of miR396 sequences in different species showed variations in the 5' portion of the miRNA, a region known to be important for miRNA activity. Analysis of different miR396 variants in Arabidopsis thaliana revealed that they have an enhanced activity toward GRF transcription factors. The interaction between the GRF target site and miR396 has a bulge between positions 7 and 8 of the miRNA. Our data indicate that such bulge modulates the strength of the miR396-mediated repression and that this modulation is essential to shape the precise spatio-temporal pattern of GRF2 expression. The results show that ancient miRNAs can regulate conserved targets with varied efficiency in different species, and we further propose that they could acquire new targets whose control might also be biologically relevant.