RESUMO
Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks.
Assuntos
Neoplasias Ovarianas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de Sinais , Feminino , Humanos , Armazenamento e Recuperação da Informação , Modelos Moleculares , Mutação Puntual , Proteínas Quinases/química , SoftwareRESUMO
Light makes carbon fixation possible, allowing plant and animal life on Earth. We have previously shown that light regulates alternative splicing in plants. Light initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing of a subset of Arabidopsis thaliana transcripts. Here, we show that light promotes RNA polymerase II (Pol II) elongation in the affected genes, whereas in darkness, elongation is lower. These changes in transcription are consistent with elongation causing the observed changes in alternative splicing, as revealed by different drug treatments and genetic evidence. The light control of splicing and elongation is abolished in an Arabidopsis mutant defective in the transcription factor IIS (TFIIS). We report that the chloroplast control of nuclear alternative splicing in plants responds to the kinetic coupling mechanism found in mammalian cells, providing unique evidence that coupling is important for a whole organism to respond to environmental cues.
Assuntos
Processamento Alternativo/efeitos da radiação , Arabidopsis/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Plantas Geneticamente Modificadas/efeitos da radiação , RNA de Plantas/efeitos da radiação , Elongação da Transcrição Genética/efeitos da radiação , Acetilação , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Escuridão , Histonas/genética , Histonas/metabolismo , Cinética , Mutação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA de Plantas/biossíntese , RNA de Plantas/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Accurate characterisation of splice junctions (SJs) as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA-seq data, and enables detailed investigations of transcriptional and post-transcriptional regulation. Using novel computational methods and a combination of PacBio Iso-seq and Illumina short-read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset from the European 2-row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the SJs and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared with an earlier version, BaRTv1.0. The accuracy of transcript level quantification, SJs and transcript start and end sites have been validated extensively using parallel technologies and analysis, including high-resolution reverse transcriptase-polymerase chain reaction and 5'-RACE. BaRTv2.18 contains 39 434 genes and 148 260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high-quality resource for advanced transcriptomic analyses, including both transcriptional and post-transcriptional regulation, with exceptional resolution and precision.
Assuntos
Hordeum , Transcriptoma , Perfilação da Expressão Gênica/métodos , Hordeum/genética , RNA-Seq , Análise de Sequência de RNA/métodos , Transcriptoma/genéticaRESUMO
Nonsense-mediated RNA decay (NMD) is an RNA control mechanism that has also been implicated in the broader regulation of gene expression. Nevertheless, a role for NMD in genome regulation has not yet been fully assessed, partially because NMD inactivation is lethal in many organisms. Here, we performed an in-depth comparative analysis of Arabidopsis (Arabidopsis thaliana) mutants lacking the NMD-related proteins UPF3, UPF1, and SMG7. We found different impacts of these proteins on NMD and the Arabidopsis transcriptome, with UPF1 having the biggest effect. Transcriptome assembly in UPF1-null plants revealed genome-wide changes in alternative splicing, suggesting that UPF1 functions in splicing. The inactivation of UPF1 led to translational repression, as manifested by a global shift in mRNAs from polysomes to monosomes and the downregulation of genes involved in translation and ribosome biogenesis. Despite these global changes, NMD targets and mRNAs expressed at low levels with short half-lives were enriched in the polysomes of upf1 mutants, indicating that UPF1/NMD suppresses the translation of aberrant RNAs. Particularly striking was an increase in the translation of TIR domain-containing, nucleotide binding, leucine-rich repeat (TNL) immune receptors. The regulation of TNLs via UPF1/NMD-mediated mRNA stability and translational derepression offers a dynamic mechanism for the rapid activation of TNLs in response to pathogen attack.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/metabolismo , Processamento Alternativo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Regulação da Expressão Gênica de Plantas , Mutação , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , RNA Helicases/genéticaRESUMO
Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase-substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine-threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase ß3-αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/metabolismo , Catálise , Linhagem Celular , Humanos , Mutagênese/genética , Mutagênese/fisiologia , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Quinases Ativadas por p21/genéticaRESUMO
Fasting rapidly (≤ 6 h) activates mitochondrial biogenic pathways in rodent muscle, an effect that is absent in human muscle following prolonged (10-72 h) fasting. We tested the hypotheses that fasting-induced changes in human muscle occur shortly after food withdrawal and are modulated by whole-body energetic stress. Vastus lateralis biopsies were obtained from ten healthy males before, during (4 h), and after (8 h) two supervised fasts performed with (FAST+EX) or without (FAST) 2 h of arm ergometer exercise (~ 400 kcal of added energy expenditure). PGC-1α mRNA (primary outcome measure) was non-significantly reduced (p = 0.065 [ηp2 = 0.14]) whereas PGC-1α protein decreased (main effect of time: p < 0.01) during both FAST and FAST+EX. P53 acetylation increased in both conditions (main effect of time: p < 0.01) whereas ACC and SIRT1 phosphorylation were non-significantly decreased (both p < 0.06 [ηp2 = 0.15]). Fasting-induced increases in NFE2L2 and NRF1 protein were observed (main effects of time: p < 0.03), though TFAM and COXIV protein remained unchanged (p > 0.05). Elevating whole-body energetic stress blunted the increase in p53 mRNA, which was apparent during FAST only (condition × time interaction: p = 0.04). Select autophagy/mitophagy regulators (LC3BI, LC3BII, BNIP3) were non-significantly reduced at the protein level (p ≤ 0.09 [ηp2 > 0.13]) but the LC3II:I ratio was unchanged (p > 0.05). PDK4 mRNA (p < 0.01) and intramuscular triglyceride content in type IIA fibers (p = 0.04) increased similarly during both conditions. Taken together, human skeletal muscle signaling, mRNA/protein expression, and substrate storage appear to be unaffected by whole-body energetic stress during the initial hours of fasting.
Assuntos
Restrição Calórica , Metabolismo Energético , Exercício Físico , Jejum/metabolismo , Mitocôndrias Musculares/metabolismo , Contração Muscular , Músculo Quadríceps/metabolismo , Acetilação , Adaptação Fisiológica , Adolescente , Adulto , Estudos Cross-Over , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Mitocôndrias Musculares/genética , Fator 1 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Distribuição Aleatória , Fatores de Tempo , Adulto JovemRESUMO
Leucine-rich pentatricopeptide repeat motif-containing protein (LRP130) is implicated in the control of mitochondrial gene expression and oxidative phosphorylation in the liver, partly due to its interaction with peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α). To investigate LRP130's role in healthy human skeletal muscle, we examined LRP130's fiber-type distribution and subcellular localization (n = 6), as well as LRP130's relationship with PGC-1α protein and citrate synthase (CS) maximal activity (n = 33) in vastus lateralis samples obtained from young males. The impact of an acute bout of exercise (endurance [END] and sprint interval training [SIT]) and fasting (8 h) on LRP130 and PGC-1α expression was also determined (n = 10). LRP130 protein content paralleled fiber-specific succinate dehydrogenase activity (I > IIA) and strongly correlated with the mitochondrially localized protein apoptosis-inducing factor in type I (r = 0.75) and type IIA (r = 0.85) fibers. Whole-muscle LRP130 protein content was positively related to PGC-1α protein (r = 0.49, p < 0.01) and CS maximal activity (r = 0.42, p < 0.01). LRP130 mRNA expression was unaltered (p > 0.05) following exercise, despite ~ 6.6- and ~ 3.8-fold increases (p < 0.01) in PGC-1α mRNA expression after END and SIT, respectively. Although unchanged at the group level (p > 0.05), moderate-to-strong positive correlations were apparent between individual changes in LRP130 and PGC-1α expression at the mRNA (r = 0.63, p < 0.05) and protein (r = 0.59, p = 0.07) level in response to fasting. Our findings support a potential role for LRP130 in the maintenance of basal mitochondrial phenotype in human skeletal muscle. LRP130's importance for mitochondrial remodeling in exercised and fasted human skeletal muscle requires further investigation.
Assuntos
Exercício Físico/fisiologia , Jejum/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Neoplasias/metabolismo , Descanso/fisiologia , Adulto , Animais , Apoptose/fisiologia , Citrato (si)-Sintase/metabolismo , Jejum/fisiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Musculares/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
PURPOSE: To examine the relationship between changes in nuclear factor erythroid 2-related factor 2 (Nrf2) expression and markers of mitochondrial biogenesis in acutely and chronically exercised human skeletal muscle. METHODS: The impact of acute submaximal endurance (END) and supramaximal interval (Tabata) cycling on the upregulation of Nrf2 (and its downstream targets), nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (TFAM) mRNA expression was examined in healthy young males (n = 10). The relationship between changes in citrate synthase (CS) maximal activity and the protein content of Nrf2, heme oxygenase 1 (HO-1), NRF-1, and TFAM was also investigated following 4 weeks of Tabata in a separate group of males (n = 21). RESULTS: Nrf2, NRF-1, and HO-1 mRNA expression increased after acute exercise (p < 0.05), whereas the increase in superoxide dismutase 2 (SOD2) mRNA expression approached significance (p = 0.08). Four weeks of Tabata increased CS activity and Nrf2, NRF-1, and TFAM protein content (p < 0.05), but decreased HO-1 protein content (p < 0.05). Training-induced changes in Nrf2 protein were strongly correlated with NRF-1 (r = 0.63, p < 0.01). When comparing protein content changes between individuals with the largest (HI: + 23%) and smallest (LO: - 1%) observed changes in CS activity (n = 8 each), increases in Nrf2 and TFAM protein content were apparent in the HI group only (p < 0.02) with medium-to-large effect sizes for between-group differences in changes in Nrf2 (ηp2=0.15) and TFAM (ηp2 = 0.12) protein content. CONCLUSION: Altogether, our findings support a potential role for Nrf2 in exercise-induced mitochondrial biogenesis in human skeletal muscle.
Assuntos
Músculo Esquelético/metabolismo , Fator 2 Relacionado a NF-E2/genética , Biogênese de Organelas , Condicionamento Físico Humano/métodos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Masculino , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adulto JovemRESUMO
BACKGROUND: The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. RESULTS: A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts - BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427-433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20-28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5' and 3' UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. CONCLUSION: A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.
Assuntos
Perfilação da Expressão Gênica/métodos , Hordeum/genética , Proteínas de Plantas/genética , Processamento Alternativo , Bases de Dados Genéticas , Regulação da Expressão Gênica de Plantas , Análise de Sequência de RNA , Sequenciamento do ExomaRESUMO
The ability to sense and respond to sugar signals allows plants to cope with environmental and metabolic changes by adjusting growth and development accordingly. We previously reported that the SR45 splicing factor negatively regulates glucose signaling during early seedling development in Arabidopsis thaliana Here, we show that under glucose-fed conditions, the Arabidopsis sr45-1 loss-of-function mutant contains higher amounts of the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1) despite unaffected SnRK1 transcript levels. In agreement, marker genes for SnRK1 activity are upregulated in sr45-1 plants, and the glucose hypersensitivity of sr45-1 is attenuated by disruption of the SnRK1 gene. Using a high-resolution RT-PCR panel, we found that the sr45-1 mutation broadly targets alternative splicing in vivo, including that of the SR45 pre-mRNA itself. Importantly, the enhanced SnRK1 levels in sr45-1 are suppressed by a proteasome inhibitor, indicating that SR45 promotes targeting of the SnRK1 protein for proteasomal destruction. Finally, we demonstrate that SR45 regulates alternative splicing of the Arabidopsis 5PTase13 gene, which encodes an inositol polyphosphate 5-phosphatase previously shown to interact with and regulate the stability of SnRK1 in vitro, thus providing a mechanistic link between SR45 function and the modulation of degradation of the SnRK1 energy sensor in response to sugars.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estabilidade Proteica , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
NEW FINDINGS: What is the central question of this study? Are individual changes in exercise-induced mRNA expression repeatable (i.e. representative of the true response to exercise rather than random error)? What is the main finding and its importance? Exercise-induced changes in mRNA expression are not repeatable even under identical experimental conditions, thereby challenging the use of mRNA expression as a biomarker of adaptive potential and/or individual responsiveness to exercise. ABSTRACT: It remains unknown if (1) the observed change in mRNA expression reflects an individual's true response to exercise or random (technical and/or biological) error, and (2) the individual responsiveness to exercise is protocol-specific. We examined the repeatability of skeletal muscle PGC-1α, PDK4, NRF-1, VEGF-A, HSP72 and p53 mRNA expression following two identical endurance exercise (END) bouts (END-1, END-2; 30 min of cycling at 65% of peak work rate (WRpeak ), n = 11) and inter-individual variability in PGC-1α and PDK4 mRNA expression following END and sprint interval training (SIT; 8 × 20 s cycling intervals at â¼170% WRpeak , n = 10) in active young males. The repeatability of key gene analysis steps (RNA extraction, reverse transcription, qPCR) and within-sample fibre-type distribution (n = 8) was also determined to examine potential sources of technical error in our analyses. Despite highly repeatable exercise bout characteristics (work rate, heart rate, blood lactate; ICC > 0.71; CV < 10%; r > 0.85, P < 0.01), gene analysis steps (ICC > 0.73; CV < 24%; r > 0.75, P < 0.01), and similar group-level changes in mRNA expression, individual changes in PGC-1α, PDK4, VEGF-A and p53 mRNA expression were not repeatable (ICC < 0.22; CV > 20%; r < 0.21). Fibre-type distribution in two portions of the same muscle biopsy was highly variable and not significantly related (ICC = 0.39; CV = 26%; r = 0.37, P = 0.37). Since individual changes in mRNA expression following identical exercise bouts were not repeatable, inferences regarding individual responsiveness to END or SIT were not made. Substantial random error exists in changes in mRNA expression following acute exercise, thereby challenging the use of mRNA expression for analysing individual responsiveness to exercise.
Assuntos
Exercício Físico/fisiologia , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo , Adulto , Treinamento Intervalado de Alta Intensidade/métodos , Humanos , Masculino , Reação em Cadeia da Polimerase/métodos , Adulto JovemRESUMO
One of the ways in which plants can respond to temperature is via alternative splicing (AS). Previous work showed that temperature changes affected the splicing of several circadian clock gene transcripts. Here, we investigated the role of RNA-binding splicing factors (SFs) in temperature-sensitive AS of the clock gene LATE ELONGATED HYPOCOTYL (LHY). We characterized, in wild type plants, temperature-associated isoform switching and expression patterns for SF transcripts from a high-resolution temperature and time series RNA-seq experiment. In addition, we employed quantitative RT-PCR of SF mutant plants to explore the role of the SFs in cooling-associated AS of LHY. We show that the splicing and expression of several SFs responds sufficiently, rapidly, and sensitively to temperature changes to contribute to the splicing of the 5'UTR of LHY. Moreover, the choice of splice site in LHY was altered in some SF mutants. The splicing of the 5'UTR region of LHY has characteristics of a molecular thermostat, where the ratio of transcript isoforms is sensitive to temperature changes as modest as 2 °C and is scalable over a wide dynamic range of temperature. Our work provides novel insight into SF-mediated coupling of the perception of temperature to post-transcriptional regulation of the clock.
Assuntos
Processamento Alternativo , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Arabidopsis/fisiologia , Ritmo Circadiano/genética , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica de Plantas , Isoformas de RNA/genética , Isoformas de RNA/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Temperatura , Fatores de Transcrição/fisiologiaRESUMO
Transcript annotation in plant databases is incomplete and often inaccurate, leading to misinterpretation. As more and more RNA-seq data are generated, plant scientists need to be aware of potential pitfalls and understand the nature and impact of specific alternative splicing transcripts on protein production. A primary area of concern and the topic of this article is the (mis)annotation of open reading frames and premature termination codons. The basic message is that to adequately address expression and functions of transcript isoforms, it is necessary to be able to predict their fate in terms of whether protein isoforms are generated or specific transcripts are unproductive or degraded.
Assuntos
Processamento Alternativo , Proteínas de Plantas/genética , Plantas/genética , Biossíntese de Proteínas/genética , Modelos Genéticos , Fases de Leitura Aberta/genética , Isoformas de Proteínas/genética , Estabilidade de RNA , RNA Mensageiro/genéticaRESUMO
BACKGROUND: The changing climate is altering timing of key fruit ripening processes and increasing the occurrence of fruit defects. To improve our understanding of the genetic control of raspberry fruit development an enhanced genetic linkage map was developed and used to examine ripening phenotypic data. RESULTS: In this study we developed an enhanced genetic linkage map for the raspberry cvs. Glen Moy x Latham reference mapping population using genotyping by sequencing (GbS). Alignment to a newly sequenced draft reference genome of red raspberry, cultivar (cv.) Glen Moy, identified 8019 single nucleotide polymorphisms (SNPs). After stringent filtering to take account of read coverage over all the progeny individuals, association with a single chromosome, heterozygosity and marker regression mapping, 2348 high confidence SNPs were retained and integrated with an existing raspberry genetic map. The linkage map contained many more SNPs segregating in Latham than in Glen Moy. This caused difficulties in quantitative trait loci (QTL) mapping with standard software and a novel analysis based on a hidden Markov model was used to improve the mapping. QTL mapping using the newly generated dense genetic map not only corroborated previously identified genetic locations but also provided additional genetic elements controlling fruit ripening in raspberry. CONCLUSION: The high-density GbS map located the QTL peaks more precisely than in earlier studies, aligned the QTLs with Glen Moy genome scaffolds, narrowed the range of potential candidate genes to these regions that can be utilised in other populations or in gene expression studies to confirm their role and increased the repertoire of markers available to understand the genetic control of fruit ripening traits.
Assuntos
Frutas/genética , Ligação Genética , Organogênese Vegetal/genética , Polimorfismo de Nucleotídeo Único , Rubus/genética , Mapeamento Cromossômico , Frutas/crescimento & desenvolvimento , Locos de Características Quantitativas , Rubus/crescimento & desenvolvimentoRESUMO
The mechanisms by which poikilothermic organisms ensure that biological processes are robust to temperature changes are largely unknown. Temperature compensation, the ability of circadian rhythms to maintain a relatively constant period over the broad range of temperatures resulting from seasonal fluctuations in environmental conditions, is a defining property of circadian networks. Temperature affects the alternative splicing (AS) of several clock genes in fungi, plants, and flies, but the splicing factors that modulate these effects to ensure clock accuracy throughout the year remain to be identified. Here we show that GEMIN2, a spliceosomal small nuclear ribonucleoprotein assembly factor conserved from yeast to humans, modulates low temperature effects on a large subset of pre-mRNA splicing events. In particular, GEMIN2 controls the AS of several clock genes and attenuates the effects of temperature on the circadian period in Arabidopsis thaliana. We conclude that GEMIN2 is a key component of a posttranscriptional regulatory mechanism that ensures the appropriate acclimation of plants to daily and seasonal changes in temperature conditions.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas do Complexo SMN/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Arabidopsis/fisiologia , Proteínas de Arabidopsis/fisiologia , Ritmo Circadiano , Análise por Conglomerados , Evolução Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Teste de Complementação Genética , Estudo de Associação Genômica Ampla , Humanos , Íntrons , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/genética , Folhas de Planta/fisiologia , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas do Complexo SMN/genética , Homologia de Sequência de Aminoácidos , Spliceossomos/fisiologia , Temperatura , Transcrição GênicaRESUMO
KEY MESSAGE: QTL mapping identifies a range of underlying and unrelated genes with apparent roles in raspberry fruit ripening and softening that show characteristic developing fruit expression profiles. Fruit softening is an important agronomical trait that involves a complex interaction of plant cell processes. We have used both qualitative and quantitative scoring of fruit firmness, length, mass, and resistance to applied force to identify QTL in a raspberry mapping population. QTLs were located primarily on linkage group (LG) 3 with other significant loci on LG 1 and LG 5 and showed mostly additive effects between the two parents. The expression of key genes that underlie these QTLs with roles in cell-wall solubility, water uptake, polyamine synthesis, transcription, and cell respiration was tested across five stages of fruit development, from immature green to red ripe fruit, using real-time RT-qPCR. Gene expression patterns showed variable expression patterns across fruit development with a highly significant positive and negative correlation between genes, supporting precise regulation of expression of different cell processes throughout raspberry fruit development. Variable timing in expression was also found in some genes at different fruit development stages between soft and firm cultivars. Multiple processes have a role to play in fruit softening and this will require development of multiple marker combinations to genes that characterise raspberry fruit softening.
Assuntos
Frutas/fisiologia , Genes de Plantas , Locos de Características Quantitativas , Rubus/genética , Mapeamento Cromossômico , Regulação da Expressão Gênica de Plantas , Ligação Genética , Fenótipo , Rubus/fisiologiaRESUMO
NEW FINDINGS: What is the central question of this study? Evidence from cellular and animal models suggests that SIRT3 is involved in regulating aerobic ATP production. Thus, we investigated whether changes in fatty acid and oxidative metabolism known to accompany fasting and exercise occur in association with changes in SIRT3 mitochondrial localization and expression in human skeletal muscle. What is the main finding and its importance? We find that 48 h of fasting and acute endurance exercise decrease SIRT3 mRNA expression but do not alter SIRT3 mitochondrial localization despite marked increases in fatty acid oxidation. This suggests that SIRT3 activity is not regulated by changes in mitochondrial localization in response to cellular energy stress in human skeletal muscle. The present study examined SIRT3 expression and SIRT3 mitochondrial localization in response to acute exercise and short-term fasting in human skeletal muscle. Experiment 1 involved eight healthy men (age, 21.4 ± 2.8 years; peak O2 uptake, 47.1 ± 11.8 ml min(-1) kg(-1) ) who performed a single bout of exercise at â¼55% of peak aerobic work rate for 1 h. Muscle biopsies were obtained at rest (Rest), immediately after exercise (EX-0) and 3 h postexercise (EX-3). Experiment 2 involved 10 healthy men (age, 22.0 ± 1.5 years; peak O2 uptake, 46.9 ± 6.0 ml min−1 kg−1) who underwent a 48 h fast, with muscle biopsies collected 1 h postprandial (Fed) and after 48 h of fasting (Fast). Mitochondrial respiration was measured using high-resolution respirometry in permeabilized muscle fibre bundles to assess substrate oxidation. Whole body fat oxidation increased after both exercise (Rest, 0.96 ± 0.32 kcal min(-1) ; Exercise, 5.66 ± 1.97 kcal min(-1) ; P < 0.001) and fasting (Fed, 0.87 ± 0.51 kcal min(-1) ; Fast, 1.30 ± 0.37 kcal min(-1) , P < 0.05). SIRT3 gene expression decreased (P < 0.05) after both exercise (-8%) and fasting (-19%); however, SIRT3 whole muscle protein content was unaltered after fasting. No changes were observed in SIRT3 mitochondrial localization following either exercise or fasting. Fasting also decreased the Vmax of glutamate [80 ± 43 versus 50 ± 21 pmol s(-1) (mg dry weight)(-1) ; P < 0.05]. These findings suggest that SIRT3 does not appear to be regulated by changes in mitochondrial localization at the time points measured in the present study in response to cellular energy stress in human skeletal muscle.
Assuntos
Exercício Físico/fisiologia , Jejum/fisiologia , Expressão Gênica/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Sirtuína 3/genética , Sirtuína 3/metabolismo , Adulto , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Oxirredução , Descanso/fisiologia , Adulto JovemRESUMO
Circadian rhythms allow organisms to time biological processes to the most appropriate phases of the day-night cycle. Post-transcriptional regulation is emerging as an important component of circadian networks, but the molecular mechanisms linking the circadian clock to the control of RNA processing are largely unknown. Here we show that PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5), which transfers methyl groups to arginine residues present in histones and Sm spliceosomal proteins, links the circadian clock to the control of alternative splicing in plants. Mutations in PRMT5 impair several circadian rhythms in Arabidopsis thaliana and this phenotype is caused, at least in part, by a strong alteration in alternative splicing of the core-clock gene PSEUDO RESPONSE REGULATOR 9 (PRR9). Furthermore, genome-wide studies show that PRMT5 contributes to the regulation of many pre-messenger-RNA splicing events, probably by modulating 5'-splice-site recognition. PRMT5 expression shows daily and circadian oscillations, and this contributes to the mediation of the circadian regulation of expression and alternative splicing of a subset of genes. Circadian rhythms in locomotor activity are also disrupted in dart5-1, a mutant affected in the Drosophila melanogaster PRMT5 homologue, and this is associated with alterations in splicing of the core-clock gene period and several clock-associated genes. Our results demonstrate a key role for PRMT5 in the regulation of alternative splicing and indicate that the interplay between the circadian clock and the regulation of alternative splicing by PRMT5 constitutes a common mechanism that helps organisms to synchronize physiological processes with daily changes in environmental conditions.