Oncode Institute, Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, Utrecht, The Netherlands.
Nature. 2021 Sep;597(7877):561-565. doi: 10.1038/s41586-021-03887-4. Epub 2021 Sep 8.
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 genomes, epigenomes and transcriptomes of single cells. However, despite recent progress in detecting proteins by mass spectrometry with single-cell resolution, it remains a major challenge to measure translation in individual cells. Here, building on existing protocols, 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.
单细胞测序方法使我们能够深入分析广泛生物体内的细胞类型和细胞状态的多样性。这些工具主要集中在对单个细胞的基因组、表观基因组和转录组进行测序。然而，尽管近年来通过单细胞分辨率的质谱法在检测蛋白质方面取得了进展，但在单个细胞中测量翻译仍然是一个主要挑战。在这里，我们在现有方案的基础上，大幅提高了这些测定的灵敏度，从而能够在单个细胞中进行核糖体分析。该技术与机器学习方法相结合，实现了单密码子分辨率。我们通过证明特定氨基酸的限制会导致核糖体在编码该氨基酸的一部分密码子上暂停，从而验证了这种方法。值得注意的是，这种暂停仅在与细胞周期状态相关的细胞亚群中观察到。我们在非限制条件下进一步扩展了这一现象，并在有丝分裂过程中检测到明显的 GAA 暂停。最后，我们证明了该技术在稀有原肠内分泌细胞中的适用性。这项技术为确定翻译过程对看似相同的细胞之间显著多样性的贡献迈出了第一步。
