FXR1P相互作用RNA的研究

The Study of FXR1P-Interacting mRNA

目的:本研究利用酵母三杂交系统从人脑海马回cDNA文库中筛选FXR1P的靶RNA,以进一步阐明FXR1基因的功能。方法:将表达FXR1P全长的质粒pYESTrp3/FXR1转化酵母菌株L40ura3/pHybLex/Zeo-MS2,检测毒性和自激活性;应用酵母三杂交技术从人脑海马回pRH3′-cDNA文库中筛选FXR1P的相互作用RNA;分离初步筛选的结果,再次转化含有诱饵质粒的融合菌株L40ura3/pHybLex/Zeo-MS2/pYESTrp3/FXR1,重新验证阳性结果;最后对阳性结果的外源插入片段进行测序和生物信息学分析。结果:酵母三杂交的筛选得到了3个阳性结果,经过测序和同源性分析,其中一个阳性结果中的插入片段为K-ALPHA-1 mRNA的部分序列。结论:K-ALPHA-1 mRNA可能是一种新的FXR1P的靶RNA。

Objective： FXR1 （Fragile X related gene 1） is a housekeeping gene that plays an important role in the development process of nervous system and muscular system. FXR1P, the translational product of FXR1, is a kind of RNA- binding protein, but the knowledge about FXR1P＇s targeting-RNA is known litde. Therefore, the aim of this study is to use yeast three hybrid system to screen FXR1P＇s target RNA from pRH3＇-cDNA library in gyrus hippocampi of human brain to further clarify the function of FXR1 gene. Methods： The FXR1P whole long coding vector pYESTrp3/FXR1 was introduced into host strain L40 ura3/pHybLex/Zeo-MS2. Then the toxicity and self-activation of pYESTrp3/FXR1 were tested. The human brain pRH3＇-cDNA library was screened by yeast transformation. The plasmids were extracted from the obtained positive yeast clones and reintroduced into the L40 ura3/pHybLex/Zeo-MS2/pYESTrp3/FXR1 one by one so as to validate the RNA-protein interactions. The inserted fragments of identified positive clones were sequenced and analyzed with BLAST in NCBI. Results： Three positive results were obtained through yeast three hybrid screening, in one of which, the inserted fragments was the partial sequence of K-ALPHA-1 mRNA by sequencing and BLAST analysis. Conclusions： K-ALPHA-1 mRNA is likely to be a new target RNA of FXR1P ,which maybe plays a role in the genesis of Fragile X syndrome.