Raponi Mitch, Arndt Greg M
Department of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, NSW 2052, Australia.
Nucleic Acids Res. 2002 Jun 1;30(11):2546-54. doi: 10.1093/nar/30.11.2546.
Specific gene silencing has been demonstrated in a number of organisms by the introduction of antisense RNA. Mutagenesis of host-encoded factors has begun to unravel the mechanism of several forms of RNA-mediated gene silencing and has suggested that it may have been conserved through evolution. This has led to the identification of certain host genes, which, when mutated, abrogate this phenomenon. Conversely, the identification of other factors that, when co-expressed or overexpressed, can enhance gene inhibition is equally important for both elucidating the mechanism of this process and enhancing gene silencing in recalcitrant systems. We have taken such a dominant genetic approach to identify several host-encoded factors that dramatically enhance target gene silencing when co-expressed with antisense RNA in fission yeast. The transcription factor thi1 and, surprisingly, the ATP-dependent RNA helicase ded1 were initially shown to enhance gene silencing in this system. Additionally, screening of a Schizosaccharomyces pombe cDNA library identified four novel antisense-enhancing sequences (aes factors) all of which are homologous to genes encoding proteins with natural affinities for nucleic acids. These findings demonstrate the utility of this strategy in identifying host-encoded factors that can modulate gene silencing when co-expressed with antisense RNA and possibly other forms of gene-silencing activators.
通过导入反义RNA，已在多种生物体中证实了特异性基因沉默。对宿主编码因子进行诱变已开始揭示几种RNA介导的基因沉默形式的机制，并表明其可能在进化过程中得以保留。这导致了某些宿主基因的鉴定，这些基因发生突变时会消除这种现象。相反，鉴定出其他一些因子，当它们共表达或过表达时可以增强基因抑制作用，这对于阐明这一过程的机制以及增强顽固系统中的基因沉默同样重要。我们采用了这种显性遗传方法来鉴定几种宿主编码因子，这些因子在裂殖酵母中与反义RNA共表达时可显著增强靶基因沉默。转录因子thi1以及令人惊讶的是，ATP依赖性RNA解旋酶ded1最初显示在该系统中增强基因沉默。此外，对粟酒裂殖酵母cDNA文库的筛选鉴定出四个新的反义增强序列（aes因子），所有这些序列都与编码对核酸具有天然亲和力的蛋白质的基因同源。这些发现证明了该策略在鉴定宿主编码因子方面的实用性，这些因子与反义RNA以及可能的其他形式的基因沉默激活剂共表达时可以调节基因沉默。
