Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip)
Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip)
复制标题
DOI：
10.1093/nar/gkq024
复制
发表时间：
2010-05-01
影响因子：
14.9
通讯作者：
Ju, Jingfang
中科院分区：
文献类型：
--
作者：
Kudo, Kenji;Xi, Yaguang;Ju, Jingfang
我们已经开发了一种新的方法来系统地研究少数细胞的转录后调控。主动翻译mrna与多体相关，新合成的肽链与hsp70等分子伴侣密切相关，这些分子伴侣有助于新生多肽正确折叠成更高有序的结构。这些伴侣提供了一个锚点，将与多聚体相关的主动翻译mrna与自由mrna分离开来。亲和捕获珠被开发用于捕获与多体复合物相关的hsp70伴侣。分离的主动翻译mrna用于高通量表达谱分析。通过体外翻译系统验证了该方法的可行性，该系统具有已知的翻译调节mRNA转录物胸苷酸合成酶（TS）。我们进一步发展了以TS和p53为阳性对照的HCT-116结肠癌细胞的方法。实时qRT-PCR分析显示，5-氟尿嘧啶治疗后，TS和p53 mrna的稳态水平未发生变化。相反，两种基因的蛋白表达和多体相关mRNA水平均升高。我们的新方法从500个细胞中揭示了这些翻译率的差异。这项技术有可能在有限数量的临床标本中进行转化控制的调查。
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.