Panopoulos Panagiotis, Mauro Vincent P
Department of Neurobiology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 2008 Nov 28;283(48):33087-93. doi: 10.1074/jbc.M804904200. Epub 2008 Oct 2.
We previously showed that a 9-nucleotide sequence from the 5' leader of the Gtx homeodomain mRNA facilitates translation initiation by base pairing to 18S rRNA. These earlier studies tested the Gtx element in isolation; we now assess the physiological relevance of this element in the context of two natural mRNAs that contain this sequence in their 5' leaders, Gtx itself and FGF2 (fibroblast growth factor 2). 2'-O-Methyl-modified RNA oligonucleotides were employed to block mRNA-rRNA base pairing by targeting either the Gtx-binding site in 18S rRNA or Gtx elements in recombinant mRNAs containing the Gtx or FGF2 5' leaders linked to a reporter cistron. Studies in cell-free lysates and transfected COS-7 cells showed that translation of mRNAs containing the Gtx or FGF2 5' leaders was decreased by > 50% when oligonucleotides targeting either the rRNA or mRNA were used. Specificity was demonstrated by showing that translation of the recombinant mRNAs was unaffected by control oligonucleotides. In addition, the specific oligonucleotides did not affect the translation of recombinant mRNAs in which the Gtx elements were mutated. Experiments performed using constructs containing Gtx and FGF2 5' leader and coding sequences ruled out possible effects of the reporter cistron. Furthermore, two-dimensional gel electrophoresis revealed that the oligonucleotides used in this study had little overall effect on the proteomes of cells transfected with these oligonucleotides. This study demonstrates that mRNA-rRNA base pairing affects the expression of two cellular mRNAs and describes a new approach for investigating putative mRNA-rRNA base pairing interactions in mammalian cells.
我们之前表明，来自Gtx同源异型域mRNA 5'前导序列的一个9核苷酸序列通过与18S rRNA碱基配对促进翻译起始。这些早期研究单独测试了Gtx元件；我们现在在两个天然mRNA的背景下评估该元件的生理相关性，这两个天然mRNA在其5'前导序列中包含该序列，即Gtx本身和FGF2（成纤维细胞生长因子2）。使用2'-O-甲基修饰的RNA寡核苷酸，通过靶向18S rRNA中的Gtx结合位点或含有与报告顺反子相连的Gtx或FGF2 5'前导序列的重组mRNA中的Gtx元件，来阻断mRNA-rRNA碱基配对。在无细胞裂解物和转染的COS-7细胞中的研究表明，当使用靶向rRNA或mRNA的寡核苷酸时，含有Gtx或FGF2 5'前导序列的mRNA的翻译减少了50%以上。通过表明重组mRNA的翻译不受对照寡核苷酸的影响来证明特异性。此外，特异性寡核苷酸不影响Gtx元件发生突变的重组mRNA的翻译。使用含有Gtx和FGF2 5'前导序列及编码序列的构建体进行的实验排除了报告顺反子可能产生的影响。此外，二维凝胶电泳显示，本研究中使用的寡核苷酸对用这些寡核苷酸转染的细胞的蛋白质组几乎没有总体影响。这项研究表明，mRNA-rRNA碱基配对影响两种细胞mRNA的表达，并描述了一种研究哺乳动物细胞中假定的mRNA-rRNA碱基配对相互作用的新方法。
