Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform

BackgroundThe 5′ untranslated regions of mRNA play an important role in their translation.ResultsHere, we describe the development of four methods of profiling mRNA 5′ ends using the Illumina sequencing platform; the first method utilizes SMART (Switching Mechanism At 5′ end of RNA Transcript) technology, while the second involves replacing the 5′ cap structure with RNA oligomers via ligation. The third and fourth methods are modifications of SMART, and involve enriching mRNA molecules with (nuclear transcripts) and without (mitochondrial transcripts) 5′ end cap structures, respectively. Libraries prepared using SMART technology gave more reproducible results, but the ligation method was advantageous in that it only sequenced mRNAs with a cap structure at the 5′ end.ConclusionsThese methods are suitable for global mapping of mRNA 5′ ends, both with and without cap structures, at a single molecule resolution. In addition, comparison of the present results obtained using different methods revealed the presence of abundant messenger RNAs without a cap structure.

背景：信使RNA（mRNA）的5'非翻译区（5′ untranslated regions）在其翻译过程中发挥关键作用。
结果：本研究开发了四种基于Illumina测序平台的mRNA 5'末端谱型分析方法：第一种方法采用SMART（RNA转录本5'端转换机制，Switching Mechanism At 5′ end of RNA Transcript）技术；第二种方法通过连接反应，用RNA寡核苷酸替换mRNA的5'帽子结构；第三种和第四种方法均为SMART技术的改良版本，分别用于富集带有5'帽子结构的mRNA（对应核转录本）以及不带有5'帽子结构的mRNA（对应线粒体转录本）。采用SMART技术构建的测序文库可获得更具可重复性的结果，而连接法的优势在于仅能对5'端带有帽子结构的mRNA进行测序。
结论：本研究开发的四种方法均可在单分子分辨率下，实现带有或不带有帽子结构的mRNA 5'末端的全局图谱绘制。此外，通过对比不同方法得到的实验结果，本研究发现了大量无帽子结构的信使RNA。