Sooncheol Lee^a,1,2, Botao Liu^b,1, Soohyun Lee^c,1,3, Sheng-Xiong Huang^d, Ben Shen^d,e, and Shu-Bing Qian^a,b,4

^aDivision of Nutritional Sciences and
^bGraduate Field of Genetics, Genomics and Development, Cornell University, Ithaca, NY 14853;
^cProboco Informatics, Ithaca, NY 14850;
^dDepartment of Chemistry and
^eDepartment of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458

Abstract
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.

genome wide, high throughput, leaky scanning, start codon

Footnotes
¹Sooncheol Lee, B.L., and Soohyun Lee contributed equally to this work.
²Present address: Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115.
³Present address: Center for Biomedical Informatics, Harvard Medical School, Boston, MA 02115.
⁴To whom correspondence should be addressed.

Author contributions: Sooncheol Lee, B.L., and S.-B.Q. designed research; Sooncheol Lee and B.L. performed research; S.-X.H. and B.S. contributed new reagents/analytic tools; Sooncheol Lee, Soohyun Lee, and S.-B.Q. analyzed data; and Soohyun Lee and S.-B.Q. wrote the paper.