RESUMO
Posttranslational modifications play a crucial role in regulating gene expression. Among these modifications, arginine methylation has recently attracted tremendous attention due to its role in multiple cellular functions. This review discusses the recent advances that have established arginine methylation as a major player in determining cytoplasmic messenger RNA (mRNA) fate. We specifically focus on research that implicates arginine methylation in regulating mRNA translation, decay, and RNA granule dynamics. Based on this research, we highlight a few emerging future avenues that will lead to exciting discoveries in this field.
Assuntos
Arginina/metabolismo , Citoplasma/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Regulação da Expressão Gênica , Metilação , Processamento de Proteína Pós-Traducional/genética , RNA Mensageiro/genéticaRESUMO
miR160 adjusts auxin-mediated development by post-transcriptional regulation of the auxin response factors ARF10/16/17. In tomato, knockdown of miR160 (sly-miR160) suggested that it is required for auxin-driven leaf blade outgrowth, but whether additional developmental events are adjusted by sly-miR160 is not clear. Here, the SlMIR160 genes and the genes of its SlARFs targets were edited by CRISPR/Cas9 resulting in the isolation of loss-of-function mutants. In addition, hypomorphic mutants that accumulate variable reduced levels of sly-miR160a were isolated. We found that the loss-of-function mutants in SlMIR160a (CR-slmir160a-6/7) produced only four wiry leaves, whereas the hypomorphic mutants developed leaves and flowers with graded developmental abnormalities. Phenotypic severity correlated with the upregulation of SlARF10A. Consistent with that, double mutants in SlMIR160a and SlARF10A restored leaf and flower development indicating that over-accumulation of SlARF10A underlay the developmental abnormalities exhibited in the CR-slmir160a mutants. Phenotype severity also correlated with the upregulation of the SHOOT MERISTEMLESS homolog Tomato Knotted 2, which in turn activated the transcription of the cytokinin biosynthesis genes SlIPT2 and SlIPT4. However, no change in Tomato Knotted 2 was detected in the absence of SlARF10A, suggesting that it is upregulated due to auxin signaling suppression by SlARF10A. Knockout of sly-miR160a-targeted SlARFs showed that whereas SlARF10A is indispensable for leaf blade outgrowth and floral organ patterning, the functions of SlARF16A and SlARF17 are redundant. Taken together our results suggest that sly-miR160a promotes blade outgrowth as well as leaf and leaflet initiation and floral organ development through the quantitative regulation of its major target SlARF10A.
Assuntos
Flores/genética , Flores/metabolismo , Ácidos Indolacéticos/metabolismo , MicroRNAs/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Proteínas de Arabidopsis , Sistemas CRISPR-Cas , Citocininas/genética , Citocininas/metabolismo , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Solanum lycopersicum/crescimento & desenvolvimento , MicroRNAs/fisiologia , Mutação , Fenótipo , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Regulação para CimaRESUMO
Porins form trimers in the outer membrane and help transport nutrients and waste products across the bacterial cell membrane. Porin loops are suitable candidates as display systems due to their high immunogenicity and presentation at the bacterial cell surface. In this study, Salmonella typhi porins (OmpC and OmpF) engineered with the Kennedy peptide from gp41 of HIV were characterised. The chimeric OmpC carrying the Kennedy peptide in loop7 did not trimerise, whereas the chimeric OmpF with the epitope in loop5 formed trimers and also was recognised by the antibodies in the HIV patient serum. The results suggest that chimeric S. typhi OmpF may be taken further as a potential candidate to develop as an epitope display system. Proteins 2017; 85:657-664. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Bactérias/química , Epitopos/química , Proteína gp41 do Envelope de HIV/química , Porinas/química , Salmonella typhi/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sítios de Ligação , Clonagem Molecular , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , HIV/genética , HIV/imunologia , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Soros Imunes/química , Biblioteca de Peptídeos , Porinas/genética , Porinas/imunologia , Ligação Proteica , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Salmonella typhi/metabolismo , Alinhamento de SequênciaRESUMO
Plant microRNAs play vital roles in auxin signaling via the negative regulation of auxin response factors (ARFs). Studies have shown that targeting of ARF10/16/17 by miR160 is indispensable for various aspects of development, but its functions in the model crop tomato (Solanum lycopersicum) are unknown. Here we knocked down miR160 (sly-miR160) using a short tandem target mimic (STTM160), and investigated its roles in tomato development. Northern blot analysis showed that miR160 is abundant in developing ovaries. In line with this, its down-regulation perturbed ovary patterning as indicated by the excessive elongation of the proximal ends of mutant ovaries and thinning of the placenta. Following fertilization, these morphological changes led to formation of elongated, pear-shaped fruits reminiscent of those of the tomato ovate mutant. In addition, STTM160-expressing plants displayed abnormal floral organ abscission, and produced leaves, sepals and petals with diminished blades, indicating a requirement for sly-miR160 for these auxin-mediated processes. We found that sly-miR160 depletion was always associated with the up-regulation of SlARF10A, SlARF10B and SlARF17, of which the expression of SlARF10A increased the most. Despite the sly-miR160 legitimate site of SlARF16A, its mRNA levels did not change in response to sly-miR160 down-regulation, suggesting that it may be regulated by a mechanism other than mRNA cleavage. SlARF10A and SlARF17 were previously suggested to function as inhibiting ARFs. We propose that by adjusting the expression of a group of ARF repressors, of which SlARF10A is a primary target, sly-miR160 regulates auxin-mediated ovary patterning as well as floral organ abscission and lateral organ lamina outgrowth.
Assuntos
MicroRNAs/metabolismo , Solanum lycopersicum/metabolismo , Flores/genética , Flores/metabolismo , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Solanum lycopersicum/genética , MicroRNAs/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
DICER-like 3 (DCL3) is a major player in heterochromatic 24-nucleotide (nt) small RNA (sRNA) and long microRNA (lmiRNA) biogenesis, and higher plant DCL3 mutants have been characterized from Arabidopsis thaliana and rice. Here, a tomato DCL3 (SlDCL3) mutant was generated through the use of trans-activated artificial miRNA and characterized. Constitutive trans-activation knocked down SlDCL3 levels by â¼64%, resulting in dramatically decreased 24-nt sRNA levels and a significant increase in 21- and 22-nt sRNAs. The latter was correlated with specific upregulation of SlDCL4 and SlDCL2b, which function in the biogenesis of 21- and 22-nt sRNAs, respectively. Moreover, at the majority of sRNA-generating genomic loci, an almost complete overlap between small RNA signatures of control and silenced seedlings was observed, suggesting that the reductions in 24-nt sRNAs at these loci were compensated for by biogenesis of 21- and 22-nt sRNAs from the same double-stranded RNA substrates. In addition, bioinformatic analysis and reduced expression in SlDCL3-silenced seedlings identified four novel tomato lmiRNAs, two of which were found to be developmentally regulated. Taken together, these results establish the requirement of SlDCL3 for the biogenesis of 24-nt sRNAs and lmiRNAs in tomato and suggest SlDCL4 and SlDCL2b as surrogates for SlDCL3.
Assuntos
Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Proteínas de Plantas/genética , Ribonuclease III/genética , Solanum lycopersicum/genética , Inativação Gênica , Solanum lycopersicum/metabolismo , MicroRNAs/metabolismo , Dados de Sequência Molecular , Mutação/genética , Proteínas de Plantas/metabolismo , Estrutura Secundária de Proteína , Ribonuclease III/metabolismo , Plântula/genética , Análise de Sequência de ProteínaRESUMO
DICER-like 1 (DCL1) is a major player in microRNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested development. Consequently, generation of dcl1 mutants by reverse genetics and functional analysis of DCL1 in late-developing organs are challenging. Here, these challenges were resolved through the unique use of trans-activated RNA interference. Global, as well as organ-specific tomato DCL1 (SlDCL1) silencing was induced by crossing the generated responder line (OP:SlDCL1IR) with the appropriate driver line. Constitutive trans-activation knocked down SlDCL1 levels by ~95%, resulting in severe abnormalities including post-germination growth arrest accompanied by decreased miRNA and 21-nucleotide small RNA levels, but prominently elevated levels of 22-nucleotide small RNAs. The increase in the 22-nucleotide small RNAs was correlated with specific up-regulation of SlDCL2b and SlDCL2d, which are probably involved in their biogenesis. Leaf- and flower-specific OP:SlDCL1IR trans-activation inhibited blade outgrowth, induced premature bud senescence and produced pale petals, respectively, emphasizing the importance of SlDCL1-dependent small RNAs in these processes. Together, these results establish OP:SlDCL1IR as an efficient tool for analysing processes regulated by SlDCL1-mediated gene regulation in tomato.
Assuntos
MicroRNAs/genética , Mutação/genética , Proteínas de Plantas/metabolismo , Ribonuclease III/metabolismo , Solanum lycopersicum/genética , Ativação Transcricional/genética , Sequência de Bases , Carotenoides/metabolismo , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes Reporter , MicroRNAs/metabolismo , Dados de Sequência Molecular , Fenótipo , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Interferente Pequeno/metabolismo , Plântula/genética , Homologia de Sequência de Aminoácidos , Regulação para Cima/genéticaRESUMO
The coat protein of cardamom mosaic virus (CdMV), a member of the genus Macluravirus, assembles into virus-like particles when expressed in an Escherichia coli expression system. The N and C-termini of the coat protein were engineered with the Kennedy peptide and the 2F5 and 4E10 epitopes of gp41 of HIV. The chimeric proteins reacted with sera from HIV positive persons and also stimulated secretion of cytokines by peripheral blood mononuclear cells from these persons. Thus, a system based on the coat protein of CdMV can be used to display HIV-1 antigens.
