Michel Audrey M, Ahern Anna M, Donohue Claire A, Baranov Pavel V
School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.
Proteomics. 2015 Jul;15(14):2410-6. doi: 10.1002/pmic.201400603. Epub 2015 Apr 23.
The boundaries of protein coding sequences are more difficult to define at the 5' end than at the 3' end due to potential multiple translation initiation sites (TISs). Even in the presence of phylogenetic data, the use of sequence information only may not be sufficient for the accurate identification of TISs. Traditional proteomics approaches may also fail because the N-termini of newly synthesized proteins are often processed. Thus ribosome profiling (ribo-seq), producing a snapshot of the ribosome distribution across the entire transcriptome, is an attractive experimental technique for the purpose of TIS location exploration. The GWIPS-viz (Genome Wide Information on Protein Synthesis visualized) browser (http://gwips.ucc.ie) provides free access to the genomic alignments of ribo-seq data and corresponding mRNA-seq data along with relevant annotation tracks. In this brief, we illustrate how GWIPS-viz can be used to explore the ribosome occupancy at the 5' ends of protein coding genes to assess the activity of AUG and non-AUG TISs responsible for the synthesis of proteoforms with alternative or heterogeneous N-termini. The presence of ribo-seq tracks for various organisms allows for cross-species comparison of orthologous genes and the availability of datasets from multiple laboratories permits the assessment of the technical reproducibility of the ribosome densities.
由于潜在的多个翻译起始位点(TIS),蛋白质编码序列的5'端边界比3'端更难界定。即使有系统发育数据,仅使用序列信息可能也不足以准确识别TIS。传统的蛋白质组学方法也可能失败,因为新合成蛋白质的N端常常会被加工。因此,核糖体谱分析(ribo-seq)能够呈现核糖体在整个转录组中的分布情况,是一种用于探索TIS位置的有吸引力的实验技术。GWIPS-viz(可视化蛋白质合成的全基因组信息)浏览器(http://gwips.ucc.ie)提供对ribo-seq数据和相应mRNA-seq数据的基因组比对以及相关注释轨迹的免费访问。在本简报中,我们说明了如何使用GWIPS-viz来探索蛋白质编码基因5'端的核糖体占据情况,以评估负责合成具有可变或异质N端的蛋白质变体的AUG和非AUG TIS的活性。各种生物体的ribo-seq轨迹的存在允许对直系同源基因进行跨物种比较,并且来自多个实验室的数据集的可用性允许评估核糖体密度的技术可重复性。