Simon Martin C, Marker Simone, Schmidt Helmut J
Department of Biology, University of Kaiserslautern, 67663 Kaiserslautern, Germany.
Gene Expr. 2006;13(3):167-78. doi: 10.3727/000000006783991809.
Variable antigens are large proteins located on the outer membrane of parasitic but also of free-living protists. Multigene families encoding surface antigens demonstrate an exclusive expression of proteins. The resulting presence of just one protein species on the cell surface is required for surface antigen function; therefore, the molecular mechanism of exclusive expression is of main interest. Regulation of gene expression and mechanisms establishing switching of antigens are hardly understood in any organism. Here we report on the reaction of Paramecium to the artificial knock down of surface antigen 51A expression by bacteria-mediated RNAi. This technique involves the feeding of dsRNA-producing bacteria. We analyzed different fragments of the target gene for dsRNA template regarding their specific knock down efficiency and found great differences. Treatment of Paramecia with RNAi against the 51A antigen demonstrated that although a massive amount of mRNA was present, the protein was not detected on the cell surface. Moreover, a minor abundance of 51D transcripts resulted in an exclusive presence of 51D proteins on the cell surface. This posttranscriptional regulation was confirmed by the transcript ratio (51A/51D) determined by real-time (RT) PCR of single cells. Because we were able to document unexclusive transcription also in wild-type cells our results indicate that this posttranscriptional regulation is a main factor of enabling exclusive gene expression. The comparison of serotype shifts, caused by efficient and inefficient knock down, indicates an involvement of full-length transcripts in regulation of gene expression. Thus, our study gives new insights into the mechanism of exclusive expression on the molecular level: (i) exclusive transcription does not occur, (ii) posttranscriptional regulation is a powerful factor enabling exclusive antigen expression, and (iii) surface antigen mRNA is shown to be involved in this mechanism in a regulating way.
可变抗原是位于寄生性原生生物以及自由生活的原生生物外膜上的大蛋白。编码表面抗原的多基因家族表现出蛋白质的特异性表达。细胞表面仅存在一种蛋白质对于表面抗原功能是必需的;因此,特异性表达的分子机制是主要研究兴趣所在。在任何生物体中,基因表达的调控以及建立抗原转换的机制都几乎不为人所知。在此,我们报道了草履虫对细菌介导的RNA干扰导致表面抗原51A表达人工敲低的反应。该技术涉及投喂产生双链RNA的细菌。我们分析了用于双链RNA模板的靶基因的不同片段的特异性敲低效率,发现存在很大差异。用针对51A抗原的RNA干扰处理草履虫表明,尽管存在大量mRNA,但在细胞表面未检测到该蛋白质。此外,51D转录本的少量存在导致细胞表面仅存在51D蛋白。通过单细胞实时(RT)PCR测定的转录本比率(51A/51D)证实了这种转录后调控。因为我们也能够在野生型细胞中记录到非特异性转录,所以我们的结果表明这种转录后调控是实现特异性基因表达的主要因素。由高效和低效敲低引起的血清型转变的比较表明全长转录本参与基因表达的调控。因此,我们的研究在分子水平上为特异性表达机制提供了新的见解:(i)不存在特异性转录,(ii)转录后调控是实现特异性抗原表达的有力因素,并且(iii)表面抗原mRNA以调节方式参与该机制。