Mitchell Cancer Institute, Mobile, AL 36688, USA.
Nucleic Acids Res. 2010 May;38(9):e104. doi: 10.1093/nar/gkq024. Epub 2010 Jan 31.
We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones provide an anchor with which to separate actively translating mRNAs associated with polysomes from free mRNAs. Affinity capture beads were developed to capture hsp70 chaperones associated with the polysome complexes. The isolated actively translating mRNAs were used for high-throughput expression profiling analysis. Feasibility was demonstrated using an in vitro translation system with known translationally regulated mRNA transcript thymidylate synthase (TS). We further developed the approach using HCT-116 colon cancer cells with both TS and p53 as positive controls. The steady-state levels of TS and p53 mRNAs were unaltered after 5-fluorouracil treatment as assessed by real-time qRT-PCR analysis. In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased. These differences in translational rate were revealed with our new approach from 500 cells. This technology has the potential to make investigation of translational control feasible with limited quantities of clinical specimens.
我们开发了一种新方法，可以系统地研究少量细胞中的转录后调控。正在翻译的 mRNA 与多核糖体相关联，新合成的肽链与分子伴侣（如 hsp70）密切相关，分子伴侣有助于将新生多肽折叠成更高级的结构。这些伴侣蛋白提供了一个锚点，用于将与多核糖体相关联的正在翻译的 mRNA 与游离 mRNA 分离。亲和捕获珠被开发用于捕获与多核糖体复合物相关的 hsp70 伴侣蛋白。分离的正在翻译的 mRNA 用于高通量表达谱分析。使用已知翻译调节的 mRNA 转录物胸苷酸合成酶（TS）的体外翻译系统证明了可行性。我们进一步使用 HCT-116 结肠癌细胞开发了该方法，其中 TS 和 p53 作为阳性对照。实时 qRT-PCR 分析表明，5-氟尿嘧啶处理后 TS 和 p53 mRNA 的稳态水平没有改变。相比之下，这两个基因的蛋白质表达和多核糖体相关 mRNA 水平都增加了。我们的新方法可以从 500 个细胞中揭示这些翻译率的差异。这项技术有可能使对翻译控制的研究能够使用有限数量的临床标本进行。
