Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells.
Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells.
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DOI：
10.3389/fmolb.2021.791455
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发表时间：
2021
影响因子：
5
通讯作者：
Aspden JL
中科院分区：
文献类型：
--
作者：
Douka K;Agapiou M;Birds I;Aspden JL
核糖体分析技术的发展改变了我们对 mRNA 翻译及其调控的理解。这种方法依赖于以精确方式翻译核糖体的 RNase 足迹，以生成具有核苷酸分辨率的核糖体位置的准确快照。在这里，我们测试了多种条件，这些条件有助于核糖体足迹的精确性，从而有助于核糖体分析的成功。我们发现 NaCl 浓度、RNaseI 来源、RNaseI 量和足迹温度均影响人神经母细胞瘤 SH-SY5Y 细胞中核糖体足迹的质量。当与果蝇 S2 细胞一起使用时，这些理想的足迹条件还提高了足迹质量。相同条件下的足迹在人类和黑腹果蝇细胞中产生不同的足迹大小和框架模式。我们还发现，在足迹分析之前用放线菌酮处理 S2 细胞会影响 ORF 中足迹的分布，但不会影响整体读长分布和框架模式，正如之前在其他生物体中发现的那样。我们的结果共同表明，影响核糖体足迹质量的因素有多种，并且精确足迹的性质因物种而异。
Our understanding of mRNA translation and its regulation has been transformed by the development of ribosome profiling. This approach relies upon RNase footprinting of translating ribosomes in a precise manner to generate an accurate snapshot of ribosome positions with nucleotide resolution. Here we tested a variety of conditions, which contribute to the preciseness of ribosome footprinting and therefore the success of ribosome profiling. We found that NaCl concentration, RNaseI source, RNaseI amount, and temperature of footprinting all contributed to the quality of ribosome footprinting in human neuroblastoma SH-SY5Y cells. These ideal conditions for footprinting also improved footprint quality when used with Drosophila melanogaster S2 cells. Footprinting under the same conditions generated different footprints sizes and framing patterns in human and D. melanogaster cells. We also found that treatment of S2 cells with cycloheximide prior to footprinting impacted the distribution of footprints across ORFs, without affecting overall read length distribution and framing pattern, as previously found in other organisms. Together our results indicate that a variety of factors affect ribosome footprint quality and the nature of precise footprinting varies across species.