Analysis of the molecular basis for mRNA dynamics in the nucleus using next generation sequencer and the function for the factor(s)of quality control.

核内的mRNA质量控制在很大程度上仍不清楚。为了分析核内信使核质控因子的分子功能，对核内信使核糖核酸的动态变化进行了全转录组分析。并用常规的生化和分子生物学方法对信使核糖核酸质量控制的重要性进行了探讨。用外切体的核心成分RRP45的siRNA或外切体的亚组分MTR4处理细胞，对整个转录组进行分析。将整个读取映射到人类基因组上，并比较了siRNA处理细胞和对照细胞在特定区域映射的读取数量的变化。通过RRP45或MTR4的siRNA处理，筛选出表达显著增强的基因。接下来，利用免疫沉淀和免疫染色等常规方法对人TRAMP(PAPD5-ZCCHC7-MTR4)复合体的鉴定和定位进行了分析。外切体和MTR4定位于核仁中。PAPD5和ZCCHC7定位于胞核。人TRAMP复合体的候选分子存在于核抽提物中。

The mRNA quality control in the nucleus remains largely unknown. Here, to analyze the molecular function of mRNA quality control factors in the nucleus, whole transcriptome analysis of the mRNA dynamics in the nucleus was exploited. And the importance of the mRNA quality control was also investigated using the conventional biochemical and molecular biology methods. The whole transcriptome was analyzed from the cells treated with siRNA of RRP45, a core component of exosome, or MTR4, a subcomponent of exosome. Whole reads were mapped on the human genome and the changes of the number of reads mapped on the specific region were compared between siRNA treated- and control cells. The genes whose expressions were markedly increased wereselected by the treatment of siRNA of RRP45 or MTR4. Next, the identification and localization of human TRAMP (PAPD5-ZCCHC7-MTR4) complex were analyzed using conventional methods like immunoprecipitation and immunostaining. Exosome and MTR4 were localized in the nucleolus. PAPD5 and ZCCHC7 were localized in the nucleus. The candidate molecules of human TRAMP complex were associated in the nuclear extract.