Single-cell quantification of ribosome occupancy in early mouse development.
Single-cell quantification of ribosome occupancy in early mouse development.
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DOI：
10.1038/s41586-023-06228-9
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发表时间：
2023-06
期刊：
影响因子：
64.8
通讯作者：
Cenik, Can
中科院分区：
文献类型：
--
作者：
Ozadam, Hakan;Tonn, Tori;Han, Crystal M.;Segura, Alia;Hoskins, Ian;Rao, Shilpa;Ghatpande, Vighnesh;Duc Tran;Catoe, David;Salit, Marc;Cenik, Can
翻译调控对哺乳动物早期胚胎发育至关重要。然而，以前的研究仅限于批量测量，排除了精确确定的翻译调节，包括等位基因特异性分析。在这里，为了应对这一挑战，我们开发了一种新的微流体等速电泳（ITP）方法，命名为通过ITP（Ribo-ITP）的核糖体分析，并在小鼠早期发育过程中表征了单个卵母细胞和胚胎的翻译。我们确定差异翻译效率作为一个关键机制，调节基因参与中心体组织和N6-甲基腺苷修饰的RNA。据我们所知，我们的高覆盖率测量使早期发育中等位基因特异性核糖体参与的第一次分析成为可能。这些导致了合子RNA与核糖体的阶段特异性差异接合的发现，并降低了表现出等位基因偏向表达的转录本的翻译效率。通过将我们的测量结果与蛋白质组学数据相结合，我们发现，核糖体在萌发囊泡期卵母细胞中的占有率是受精卵中蛋白质丰度的主要决定因素。Ribo-ITP方法将通过从包括单细胞在内的超低输入样本中提供高覆盖率和高分辨率的核糖体占有率测量来实现众多应用。单细胞核糖体分析方法可以提供早期发育中等位基因特异性核糖体接合水平的数据。
Translation regulation is critical for early mammalian embryonic development. However, previous studies had been restricted to bulk measurements, precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells. A single-cell ribosome profiling method can provide data at the level of allele-specific ribosome engagement in early development.
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