Development of SUPPA for alternative splicing analysis from RNA-seq in plants across multiple conditions

开发 SUPPA，用于多种条件下植物中 RNA-seq 的选择性剪接分析

• 批准号: BB/N022807/1
• 负责人: John Brown
• 金额: $ 11.68万
• 依托单位: University of Dundee
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN022807%2F1
• 关键词: Development; SUPPA; alternative; splicing; analysis

Genes are the repositories of hereditary information and proteins are the machines that carry out the functions of living cells. The term 'gene expression' usually refers to the process by which a gene gives rise to a protein. In eukaryotes, gene expression is complex and when protein-coding genes are expressed, the DNA sequence is first copied into a precursor messenger RNA (pre-mRNA) by transcription. The pre-mRNA undergoes several processing steps to form mature messenger RNAs (mRNAs) which direct synthesis of the corresponding protein (translation). An extremely important RNA processing step called alternative splicing (AS) generates different mRNA transcripts (i.e. isoforms) from the same gene and thereby modulates transcript and protein levels and functions. The majority of protein-coding genes undergo AS and the relative amounts of AS isoforms changes dynamically as cells and organisms develop and grow. High-throughput methods such as RNA sequencing (RNA-seq) are now capable of generating data on tens of thousands of transcripts from cells or particular stages of development or different conditions. To be able to analyse the dynamic changes in transcripts and AS, and to understand how this is regulated, we need computational tools that will allow the accurate measurement of these different mRNA transcript isoforms from these large datasets. The tool which we will develop here will enable the high resolution analysis of dynamic changes in gene expression at the individual transcript and AS event level. Being able to distinguish the abundance of different transcript isoforms is important because one of the main approaches scientists use to associate genes with functions is to monitor gene expression: i.e. where and when genes are switched on or off, and at what level. The RNA-seq technology and programmes to analyse the data are continually being improved. Significant recent advances are the release of computational programmes that can quantify transcript isoform abundance (e.g. Sailfish, Salmon, Kallisto) and can generate measures of AS (e.g. SUPPA) from large datasets very quickly. We have been using these programmes to analyse RNA-seq data from Arabidopsis. We also have an excellent experimental system which allows us to validate the RNA-seq results. Detailed comparisons have helped us to identify a number of discrepancies and issues with the SUPPA programme where it does not accurately report on, for example, complex AS events. We have taken initial steps in improving SUPPA with good success and here we aim to modify SUPPA in a number of ways so that it 1) accurately measures AS, and 2) can be applied to experiments with multiple conditions such as time-course series. The significance of this is that it will allow clustering of AS responses, correlation of AS indices with gene expression, and the building of splicing networks to understand the regulation of AS. Research groups in the UK and around the world use RNA-seq to analyse gene expression in plants and animals. Currently there are many limitations in quantifying transcripts and AS in a dynamic way - the improved version of SUPPA will deliver this function. It will be relevant not only to Arabidopsis but also to other plant and crop species, and to animal and human studies.

基因是遗传信息的储存库，蛋白质是执行活细胞功能的机器。术语“基因表达”通常指基因产生蛋白质的过程。在真核生物中，基因表达是复杂的，并且当蛋白质编码基因表达时，DNA序列首先通过转录复制成前体信使RNA（pre-mRNA）。前mRNA经历几个加工步骤以形成成熟的信使RNA（mRNA），其指导相应蛋白质的合成（翻译）。一个非常重要的RNA加工步骤称为选择性剪接（AS）从同一基因产生不同的mRNA转录本（即同种型），从而调节转录本和蛋白质水平和功能。大多数蛋白质编码基因经历AS，并且AS异构体的相对量随着细胞和生物体的发育和生长而动态变化。RNA测序（RNA-seq）等高通量方法现在能够生成来自细胞或特定发育阶段或不同条件的数万个转录本的数据。为了能够分析转录本和AS的动态变化，并了解这是如何调节的，我们需要计算工具，以便从这些大型数据集中准确测量这些不同的mRNA转录本亚型。我们将在这里开发的工具将使高分辨率分析的动态变化的基因表达在个人的转录本和AS事件水平。能够区分不同转录异构体的丰度是很重要的，因为科学家用来将基因与功能联系起来的主要方法之一是监测基因表达：即基因在何时何地打开或关闭，以及在什么水平上打开或关闭。用于分析数据的RNA-seq技术和程序正在不断改进。最近的重大进展是发布了计算程序，可以量化转录异构体丰度（例如旗鱼，鲑鱼，Kallisto），并可以非常快速地从大型数据集生成AS（例如SUPPA）的测量。我们一直在使用这些程序来分析来自拟南芥的RNA-seq数据。我们还有一个优秀的实验系统，使我们能够验证RNA-seq结果。详细的比较帮助我们确定了SUPPA计划的一些差异和问题，例如，它没有准确地报告复杂的AS事件。我们已经采取了初步措施，在改善SUPPA取得了良好的成功，在这里，我们的目标是修改SUPPA在许多方面，使它1）准确地测量AS，2）可以应用于实验与多种条件，如时间过程系列。这一点的意义在于，它将允许AS反应的聚类，AS指数与基因表达的相关性，以及剪接网络的构建，以了解AS的调控。英国和世界各地的研究小组使用RNA-seq来分析植物和动物的基因表达。目前，在以动态方式量化成绩单和AS方面存在许多限制-SUPPA的改进版本将提供此功能。它不仅与拟南芥有关，而且与其他植物和作物物种以及动物和人类研究有关。

项目成果

Alternative splicing of circadian clock genes correlates with temperature in field-grown sugarcane

生物钟基因的选择性剪接与田间种植甘蔗的温度相关

• DOI: 10.1101/718890
• 发表时间: 2019
• 作者: Dantas L

Rapid and dynamic alternative splicing impacts the Arabidopsis cold response transcriptome

快速动态的选择性剪接影响拟南芥冷反应转录组

• DOI: 10.1101/251876
• 发表时间: 2018
• 作者: Calixto C

TSIS: an R package to infer alternative splicing isoform switches for time-series data.

• DOI: 10.1093/bioinformatics/btx411
• 发表时间: 2017-10-15
• 期刊: Bioinformatics (Oxford, England)
• 作者: Guo W;Calixto CPG;Brown JWS;Zhang R
• 通讯作者: Zhang R