RESUMO
Plants have adapted to tolerate and survive constantly changing environmental conditions by reprogramming gene expression The dynamics of the contribution of alternative splicing (AS) to stress responses are unknown. RNA-sequencing of a time-series of Arabidopsis thaliana plants exposed to cold determines the timing of significant AS changes. This shows a massive and rapid AS response with coincident waves of transcriptional and AS activity occurring in the first few hours of temperature reduction and further AS throughout the cold. In particular, hundreds of genes showed changes in expression due to rapidly occurring AS in response to cold ("early AS" genes); these included numerous novel cold-responsive transcription factors and splicing factors/RNA binding proteins regulated only by AS. The speed and sensitivity to small temperature changes of AS of some of these genes suggest that fine-tuning expression via AS pathways contributes to the thermo-plasticity of expression. Four early AS splicing regulatory genes have been shown previously to be required for freezing tolerance and acclimation; we provide evidence of a fifth gene, U2B"-LIKE Such factors likely drive cascades of AS of downstream genes that, alongside transcription, modulate transcriptome reprogramming that together govern the physiological and survival responses of plants to low temperature.
Assuntos
Processamento Alternativo/genética , Arabidopsis/genética , Transcriptoma/genética , Temperatura Baixa , Regulação da Expressão Gênica de Plantas/genéticaRESUMO
RNA-sequencing (RNA-seq) analysis of gene expression and alternative splicing should be routine and robust but is often a bottleneck for biologists because of different and complex analysis programs and reliance on specialized bioinformatics skills. We have developed the '3D RNA-seq' App, an R shiny App and web-based pipeline for the comprehensive analysis of RNA-seq data from any organism. It represents an easy-to-use, flexible and powerful tool for analysis of both gene and transcript-level gene expression to identify differential gene/transcript expression, differential alternative splicing and differential transcript usage (3D) as well as isoform switching from RNA-seq data. 3D RNA-seq integrates state-of-the-art differential expression analysis tools and adopts best practice for RNA-seq analysis. The program is designed to be run by biologists with minimal bioinformatics experience (or by bioinformaticians) allowing lab scientists to analyse their RNA-seq data. It achieves this by operating through a user-friendly graphical interface which automates the data flow through the programs in the pipeline. The comprehensive analysis performed by 3D RNA-seq is extremely rapid and accurate, can handle complex experimental designs, allows user setting of statistical parameters, visualizes the results through graphics and tables, and generates publication quality figures such as heat-maps, expression profiles and GO enrichment plots. The utility of 3D RNA-seq is illustrated by analysis of data from a time-series of cold-treated Arabidopsis plants and from dexamethasone-treated male and female mouse cortex and hypothalamus data identifying dexamethasone-induced sex- and brain region-specific differential gene expression and alternative splicing.
Assuntos
Processamento Alternativo , Arabidopsis/metabolismo , Córtex Cerebelar/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipotálamo/metabolismo , RNA-Seq/métodos , RNA/genética , Animais , Arabidopsis/efeitos dos fármacos , Córtex Cerebelar/efeitos dos fármacos , Temperatura Baixa , Biologia Computacional/métodos , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Hipotálamo/efeitos dos fármacos , Camundongos , RNA/metabolismo , SoftwareRESUMO
Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.
Assuntos
Processamento Alternativo , Arabidopsis/genética , Genes de Insetos , Transcriptoma , Variação Genética , Proteômica , RNA não Traduzido , Valores de Referência , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Transcrição GênicaRESUMO
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
RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression.
Assuntos
Processamento Alternativo , Arabidopsis/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Isoformas de Proteínas/análise , RNA Mensageiro/análise , Análise de Sequência de RNA/métodos , Algoritmos , Sequência de Bases , Conjuntos de Dados como Assunto , Genes de Plantas , Splicing de RNA , Valores de Referência , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Software , TranscriptomaRESUMO
RNA-sequencing (RNA-seq) is currently the method of choice for analysis of differential gene expression. To fully exploit the wealth of data generated from genome-wide transcriptomic approaches, the initial design of the experiment is of paramount importance. Biological rhythms in nature are pervasive and are driven by endogenous gene networks collectively known as circadian clocks. Measuring circadian gene expression requires time-course experiments which take into account time-of-day factors influencing variability in expression levels. We describe here an approach for characterizing diurnal changes in expression and alternative splicing for plants undergoing cooling. The method uses inexpensive everyday laboratory equipment and utilizes an RNA-seq application (3D RNA-seq) that can handle complex experimental designs and requires little or no prior bioinformatics expertise.
Assuntos
Processamento Alternativo , Perfilação da Expressão Gênica , RNA-Seq , Projetos de Pesquisa , Análise de Sequência de RNA , TranscriptomaRESUMO
Plants re-program their gene expression when responding to changing environmental conditions. Besides differential gene expression, extensive alternative splicing (AS) of pre-mRNAs and changes in expression of long non-coding RNAs (lncRNAs) are associated with stress responses. RNA-sequencing of a diel time-series of the initial response of Arabidopsis thaliana rosettes to low temperature showed massive and rapid waves of both transcriptional and AS activity in protein-coding genes. We exploited the high diversity of transcript isoforms in AtRTD2 to examine regulation and post-transcriptional regulation of lncRNA gene expression in response to cold stress. We identified 135 lncRNA genes with cold-dependent differential expression (DE) and/or differential alternative splicing (DAS) of lncRNAs including natural antisense RNAs, sORF lncRNAs, and precursors of microRNAs (miRNAs) and trans-acting small-interfering RNAs (tasiRNAs). The high resolution (HR) of the time-series allowed the dynamics of changes in transcription and AS to be determined and identified early and adaptive transcriptional and AS changes in the cold response. Some lncRNA genes were regulated only at the level of AS and using plants grown at different temperatures and a HR time-course of the first 3 h of temperature reduction, we demonstrated that the AS of some lncRNAs is highly sensitive to small temperature changes suggesting tight regulation of expression. In particular, a splicing event in TAS1a which removed an intron that contained the miR173 processing and phased siRNAs generation sites was differentially alternatively spliced in response to cold. The cold-induced reduction of the spliced form of TAS1a and of the tasiRNAs suggests that splicing may enhance production of the siRNAs. Our results identify candidate lncRNAs that may contribute to the regulation of expression that determines the physiological processes essential for acclimation and freezing tolerance.