Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2012 Sep 11;109(37):E2424-32. doi: 10.1073/pnas.1207846109. Epub 2012 Aug 27.
Understanding translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. The recently developed ribosome-profiling technique enables global translation analysis, providing a wealth of information about both the position and the density of ribosomes on mRNAs. Here we present an approach, global translation initiation sequencing, applying in parallel the ribosome E-site translation inhibitors lactimidomycin and cycloheximide to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. This approach provides a view of alternative translation initiation in mammalian cells with single-nucleotide resolution. Systemic analysis of TIS positions supports the ribosome linear-scanning mechanism in TIS selection. The alternative TIS positions and the associated ORFs identified by global translation initiation sequencing are conserved between human and mouse cells, implying physiological significance of alternative translation. Our study establishes a practical platform for uncovering the hidden coding potential of the transcriptome and offers a greater understanding of the complexity of translation initiation.
理解基因表达中的翻译调控依赖于在整个转录组中精确和全面地确定翻译起始位点 (TIS)。最近开发的核糖体分析技术能够进行全局翻译分析，提供大量关于 mRNA 上核糖体位置和密度的信息。在这里，我们提出了一种方法，即全局翻译起始测序，同时应用核糖体 E 位翻译抑制剂乳酰胺和环己酰亚胺来实现全基因组范围内起始和延伸事件的同时检测。这种方法以单核苷酸分辨率提供了哺乳动物细胞中替代翻译起始的视图。TIS 位置的系统分析支持 TIS 选择中的核糖体线性扫描机制。通过全局翻译起始测序确定的替代 TIS 位置和相关的 ORF 在人和小鼠细胞之间是保守的，这暗示了替代翻译的生理意义。我们的研究为揭示转录组的隐藏编码潜力建立了一个实用的平台，并提供了对翻译起始复杂性的更深入理解。
