Poly A- Transcripts Expressed in HeLa Cells

BackgroundTranscripts expressed in eukaryotes are classified as poly A+ transcripts or poly A- transcripts based on the presence or absence of the 3�� poly A tail. Most transcripts identified so far are poly A+ transcripts, whereas the poly A- transcripts remain largely unknown.Methodology/Principal FindingsWe developed the TRD (Total RNA Detection) system for transcript identification. The system detects the transcripts through the following steps: 1) depleting the abundant ribosomal and small-size transcripts; 2) synthesizing cDNA without regard to the status of the 3�� poly A tail; 3) applying the 454 sequencing technology for massive 3�� EST collection from the cDNA; and 4) determining the genome origins of the detected transcripts by mapping the sequences to the human genome reference sequences. Using this system, we characterized the cytoplasmic transcripts from HeLa cells. Of the 13,467 distinct 3�� ESTs analyzed, 24% are poly A-, 36% are poly A+, and 40% are bimorphic with poly A+ features but without the 3�� poly A tail. Most of the poly A- 3�� ESTs do not match known transcript sequences; they have a similar distribution pattern in the genome as the poly A+ and bimorphic 3�� ESTs, and their mapped intergenic regions are evolutionarily conserved. Experiments confirmed the authenticity of the detected poly A- transcripts.Conclusion/SignificanceOur study provides the first large-scale sequence evidence for the presence of poly A- transcripts in eukaryotes. The abundance of the poly A- transcripts highlights the need for comprehensive identification of these transcripts for decoding the transcriptome, annotating the genome and studying biological relevance of the poly A- transcripts.

【背景】真核生物中表达的转录本根据其3'端聚腺苷酸化尾（poly A tail）的有无，被划分为聚腺苷酸化阳性（poly A+）转录本与聚腺苷酸化阴性（poly A-）转录本。截至目前已鉴定的绝大多数转录本均为poly A+类型，而poly A-转录本在很大程度上仍未被充分解析。
【材料与方法/主要研究结果】我们开发了TRD（Total RNA Detection，总RNA检测）系统用于转录本鉴定。该系统通过以下步骤实现转录本检测：1）去除丰度较高的核糖体RNA与小型转录本；2）无需考虑3'端聚腺苷酸化尾的状态即可合成互补脱氧核糖核酸（cDNA）；3）借助454测序技术（454 sequencing technology）从cDNA中大规模获取3'端表达序列标签（EST，expressed sequence tag）；4）通过将序列比对至人类参考基因组序列，确定所检测到的转录本的基因组起源。利用该系统，我们对HeLa细胞的细胞质转录本进行了表征分析。在分析的13467条独特3'端EST中，24%为poly A-转录本，36%为poly A+转录本，另有40%为双态型转录本——具备poly A+特征但不携带3'端聚腺苷酸化尾。绝大多数poly A- 3'端EST未匹配已知转录本序列；它们在基因组中的分布模式与poly A+及双态型3'端EST相似，且其比对到的基因间区域在进化上具有保守性。后续实验验证了所检测到的poly A-转录本的真实性。
【结论与意义】本研究首次为真核生物中存在poly A-转录本提供了大规模序列层面的直接证据。poly A-转录本的丰富度表明，若要完整解析转录组、精准注释基因组以及深入研究poly A-转录本的生物学相关性，就必须对这类转录本开展全面的鉴定工作。