Xu P, Wen L, Benegal G, Wang X, Buck G A
Department of Microbiology and Immunology, Medical College of Virginia Campus, Box 980678, Virginia Commonwealth University, 1101 East Marshall, Rm. 5036 Sanger Hall, Richmond, VA 23298-0678, USA.
Mol Biochem Parasitol. 2001 Jan 15;112(1):39-49. doi: 10.1016/s0166-6851(00)00341-8.
Nuclear mRNAs in trypanosomatids are generated by trans-splicing. Although trans-splicing resembles cis-splicing in many ways and most of the U RNA participants have been characterized, relatively few involved proteins have been identified. Herein, we employed a yeast three-hybrid system to identify a protein, XB1, which binds to the Trypanosoma cruzi SL RNA. XB1 is a approximately 45 kDa protein which is homologous to the essential pre-mRNA-splicing factor PRP31p from Saccharomyces cerevisiae. Gel shift assays and UV cross-linking experiments with recombinant XB1 confirmed that this T. cruzi protein binds the SL RNA in vitro. The binding site of XB1 on the SL RNA was mapped to stem-loop II by deletion of the SL RNA 'bait' in the three-hybrid system. Finally, UV cross-linking SL RNA with S100 extract indicated native XB1 protein and SL RNA interaction in T. cruzi extract.
锥虫的核mRNA是通过反式剪接产生的。尽管反式剪接在许多方面类似于顺式剪接，并且大多数U RNA参与成分已得到表征，但已鉴定出的相关蛋白质相对较少。在此，我们利用酵母三杂交系统鉴定出一种与克氏锥虫SL RNA结合的蛋白质XB1。XB1是一种约45 kDa的蛋白质，与酿酒酵母中必需的前体mRNA剪接因子PRP31p同源。用重组XB1进行的凝胶迁移试验和紫外线交联实验证实，这种克氏锥虫蛋白在体外与SL RNA结合。通过在三杂交系统中缺失SL RNA“诱饵”，将XB1在SL RNA上的结合位点定位到茎环II。最后，用S100提取物进行紫外线交联SL RNA表明，天然XB1蛋白与克氏锥虫提取物中的SL RNA存在相互作用。
