Aviner Ranen, Hofmann Sarah, Elman Tamar, Shenoy Anjana, Geiger Tamar, Elkon Ran, Ehrlich Marcelo, Elroy-Stein Orna
Department of Cell Research & Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Nucleic Acids Res. 2017 Jun 2;45(10):5945-5957. doi: 10.1093/nar/gkx326.
Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase.
mRNA翻译的精确调控对于细胞正常分裂至关重要，但对于介导这一过程的因素我们却知之甚少。为了鉴定在有丝分裂期间调控翻译的mRNA结合蛋白，我们使用无偏差定量质谱法（液相色谱-串联质谱法，LC-MS/MS）分析了间期细胞和有丝分裂细胞中多核糖体的组成。我们发现，有丝分裂多核糖体富含参与RNA加工的一组蛋白质，包括可变剪接和RNA输出。为了证明这些蛋白确实可能是翻译的调节因子，我们以不均一核核糖核蛋白C（hnRNP C）为例进行研究，并证实它在有丝分裂期间被募集到延伸中的核糖体上。然后，我们结合使用脉冲式稳定同位素标记氨基酸法（SILAC）、代谢标记法和核糖体分析技术，发现敲低hnRNP C会影响整体及转录本特异性的翻译速率，并且发现hnRNP C对于编码核糖体蛋白和翻译因子的mRNA的翻译尤为重要。综上所述，我们的结果表明多核糖体的蛋白质组学分析如何能够深入了解在各种感兴趣的细胞条件下的翻译调控，并表明hnRNP C在有丝分裂期间促进翻译机器组件的产生，以便为子细胞提供在进入G1期时高效合成蛋白质的能力。
