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Single-cell Ribo-seq reveals cell cycle-dependent translational pausing
Nature ( IF 64.8 ) Pub Date : 2021-09-08 , DOI: 10.1038/s41586-021-03887-4
Michael VanInsberghe 1 , Jeroen van den Berg 1 , Amanda Andersson-Rolf 1 , Hans Clevers 1 , Alexander van Oudenaarden 1
Affiliation  

Single-cell sequencing methods have enabled in-depth analysis of the diversity of cell types and cell states in a wide range of organisms. These tools focus predominantly on sequencing the genomes1, epigenomes2 and transcriptomes3 of single cells. However, despite recent progress in detecting proteins by mass spectrometry with single-cell resolution4, it remains a major challenge to measure translation in individual cells. Here, building on existing protocols5,6,7, we have substantially increased the sensitivity of these assays to enable ribosome profiling in single cells. Integrated with a machine learning approach, this technology achieves single-codon resolution. We validate this method by demonstrating that limitation for a particular amino acid causes ribosome pausing at a subset of the codons encoding the amino acid. Of note, this pausing is only observed in a sub-population of cells correlating to its cell cycle state. We further expand on this phenomenon in non-limiting conditions and detect pronounced GAA pausing during mitosis. Finally, we demonstrate the applicability of this technique to rare primary enteroendocrine cells. This technology provides a first step towards determining the contribution of the translational process to the remarkable diversity between seemingly identical cells.



中文翻译:

单细胞 Ribo-seq 揭示了细胞周期依赖的翻译暂停

单细胞测序方法能够深入分析各种生物体中细胞类型和细胞状态的多样性。这些工具主要侧重于对单细胞的基因组1、表观基因组2和转录组3进行测序。然而,尽管最近在单细胞分辨率4质谱法检测蛋白质方面取得了进展,但测量单个细胞中的翻译仍然是一项重大挑战。在这里,基于现有协议5,6,7,我们已经大大提高了这些检测的灵敏度,以便在单细胞中进行核糖体分析。该技术与机器学习方法相结合,可实现单密码子分辨率。我们通过证明对特定氨基酸的限制导致核糖体在编码该氨基酸的密码子子集处暂停来验证该方法。值得注意的是,这种暂停仅在与其细胞周期状态相关的细胞亚群中观察到。我们在非限制性条件下进一步扩展了这种现象,并在有丝分裂期间检测到明显的 GAA 暂停。最后,我们证明了这种技术对罕见的原发性肠内分泌细胞的适用性。该技术为确定翻译过程对看似相同细胞之间显着多样性的贡献迈出了第一步。

更新日期:2021-09-08
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