Transcriptome analysis of the model protozoan, Tetrahymena thermophila, using Deep RNA sequencing.
Transcriptome analysis of the model protozoan, Tetrahymena thermophila, using Deep RNA sequencing.
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使用深度 RNA 测序对模型原生动物嗜热四膜虫进行转录组分析
DOI：
10.1371/journal.pone.0030630
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Miao W
中科院分区：
文献类型：
--
作者：
Xiong J;Lu X;Zhou Z;Chang Y;Yuan D;Tian M;Zhou Z;Wang L;Fu C;Orias E;Miao W
纤毛虫嗜热四膜虫是细胞和分子生物学中一种经过深入研究的单细胞真核模式生物。然而，缺乏大量的嗜热四膜虫cDNA文库或大型表达序列标签（EST）数据库限制了原始基因组注释的质量。
这项RNA - seq研究描述了嗜热四膜虫在生命周期的三个主要阶段（生长、饥饿和接合）中转录组的首次深度测序分析。唯一比对上的读段覆盖了体细胞基因组中24,725个预测基因模型的96%以上。鉴定出了1000多个新的转录区域。RNA - seq的巨大动态范围使得能够检测到由该细胞精心调控的近6个数量级的可测量基因表达。RNA - seq还首次实现了对转录本非翻译区（UTRs）的预测，并对嗜热四膜虫转录组的大小进行了更新（更大）的估计：57 Mb，约占体细胞基因组的55%。我们的研究鉴定出了分布在5.2%的嗜热四膜虫基因上的近1500个可变剪接（AS）事件。这个百分比比之前基于四膜虫EST的估计值增加了两个数量级。还获得了可变剪接阶段特异性调控的证据。最后，我们的研究使我们能够完全确认基因发现程序最初预测的约26.8%的基因，纠正约三分之一基因的编码序列边界和内含子 - 外显子连接，并重新分配微阵列探针以及纠正早期的微阵列数据。
RNA - seq数据显著改善了基因组注释，并提供了嗜热四膜虫全局转录组的全面视图。据我们所知，5.2%的嗜热四膜虫基因具有可变剪接，这是单细胞真核生物中报道的具有可变剪接基因的最高百分比。因此，四膜虫成为研究可变剪接机制的一种极好的单细胞真核模式生物。
Background The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation. Methodology/Principal Findings This RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96% of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs) and an updated (larger) size estimate of the T. thermophila transcriptome: 57 Mb, or about 55% of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS) events distributed over 5.2% of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8% of the genes originally predicted by the gene finder, to correct coding sequence boundaries and intron-exon junctions for about a third, and to reassign microarray probes and correct earlier microarray data. Conclusions/Significance RNA-seq data significantly improve the genome annotation and provide a fully comprehensive view of the global transcriptome of T. thermophila. To our knowledge, 5.2% of T. thermophila genes with AS is the highest percentage of genes showing AS reported in a unicellular eukaryote. Tetrahymena thus becomes an excellent unicellular model eukaryote in which to investigate mechanisms of alternative splicing.
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