MacMorris Margaret, Brocker Chad, Blumenthal Thomas
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Denver, Colorado 80262, USA.
RNA. 2003 Jul;9(7):847-57. doi: 10.1261/rna.5480803.
Expression of a gfp transgene in the intestines of living Caenorhabditis elegans has been measured following depletion by RNAi of a variety of known splicing factors and mRNA export proteins. Reduction of most splicing factors showed only a small effect on expression of the transgene in the animal injected with dsRNA, although most of these RNAi's resulted in embryonic lethality in their offspring. In contrast, RNAi of nxf-1, the worm homolog of mammalian NXF1/TAP, a key component of the mRNA export machinery, resulted in dramatic suppression of GFP expression in the injected animals. When we tested other proteins previously reported to be involved in marking mRNAs for export, we obtained widely divergent results. Whereas RNAi of the worm REF/Aly homologs had no obvious effect, either in the injected animals or their offspring, RNAi of UAP56, reported to be the partner of REF/Aly, resulted in strong suppression of GFP expression due to nuclear retention of its mRNA. Overexpression of UAP56 also resulted in rapid loss of GFP expression and lethality at all stages of development. We conclude that UAP56 plays a key role in mRNA export in C. elegans, but that REF/Aly may not. It also appears that some RNA processing factors are required for viability (e.g., U2AF, PUF60, SRp54, SAP49, PRP8, U1-70K), whereas others are not (e.g., U2A', CstF50).
在秀丽隐杆线虫的肠道中,通过RNA干扰(RNAi)多种已知的剪接因子和mRNA输出蛋白后,已对绿色荧光蛋白(gfp)转基因的表达进行了测量。尽管大多数这些RNA干扰导致其后代胚胎致死,但减少大多数剪接因子对注射双链RNA(dsRNA)的动物中转基因的表达仅显示出很小的影响。相比之下,nxf-1(哺乳动物NXF1/TAP的线虫同源物,mRNA输出机制的关键组成部分)的RNA干扰导致注射动物中绿色荧光蛋白(GFP)表达的显著抑制。当我们测试先前报道参与标记mRNA以供输出的其他蛋白质时,我们得到了差异很大的结果。线虫REF/Aly同源物的RNA干扰在注射动物或其后代中均无明显影响,而据报道是REF/Aly伙伴的UAP56的RNA干扰则因mRNA的核滞留而导致GFP表达的强烈抑制。UAP56的过表达也导致GFP表达迅速丧失以及在发育的各个阶段出现致死性。我们得出结论,UAP56在秀丽隐杆线虫的mRNA输出中起关键作用,但REF/Aly可能并非如此。似乎一些RNA加工因子是生存所必需的(例如,U2AF、PUF60、SRp54、SAP49、PRP8、U1-70K),而其他一些则不是(例如,U2A'、CstF50)。