Lenz H J, Manno D J, Danenberg K D, Danenberg P V
Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles 90033.
J Biol Chem. 1994 Dec 16;269(50):31962-8.
The splicing activities of 5-fluorouracil (FUra)-substituted U2 and U6 small nuclear RNAs (snRNAs) were examined in an in vitro splicing system. Yeast splicing extracts were specifically depleted of endogenous U2 and U6 snRNAs by antisense oligonucleotide-directed RNase H hydrolysis. Splicing activity was recovered when the extracts were reconstituted with synthetic U2 and U6 snRNAs. However, U2 snRNA with all uracils substituted with FUra (FU2) did not restore any splicing activity. Nondenaturing gel electrophoresis showed that FU2 failed to promote the assembly of spliceosome complexes. The ability of U2 snRNA to restore splicing in U2-depleted extracts increased as FUra content decreased but was still only 60% of control activity at 25% substitution of uracils with FUra. Addition of FU2 to nondepleted extracts caused strong inhibition of splicing accompanied by increased degradation of the pre-mRNA, suggesting that FU2 forms an inactive complex with a protein splicing factor that normally binds to the pre-mRNA. FU6 restored full splicing activity to U6-depleted extracts, but at a 5-fold higher concentration than U6 snRNA. These results demonstrate that the incorporation of FUra can impair the functions of catalytic RNA molecules.
在体外剪接系统中检测了5-氟尿嘧啶（FUra）取代的U2和U6小核RNA（snRNA）的剪接活性。通过反义寡核苷酸介导的RNase H水解特异性去除酵母剪接提取物中的内源性U2和U6 snRNA。当提取物用合成的U2和U6 snRNA重建时，剪接活性得以恢复。然而，所有尿嘧啶都被FUra取代的U2 snRNA（FU2）未能恢复任何剪接活性。非变性凝胶电泳显示，FU2未能促进剪接体复合物的组装。在U2缺失的提取物中，U2 snRNA恢复剪接的能力随着FUra含量的降低而增加，但在25%的尿嘧啶被FUra取代时，其活性仍仅为对照活性的60%。向未缺失的提取物中添加FU2会导致强烈的剪接抑制，并伴随着前体mRNA降解的增加，这表明FU2与通常结合前体mRNA的蛋白质剪接因子形成了无活性的复合物。FU6可使U6缺失的提取物恢复完全的剪接活性，但所需浓度比U6 snRNA高5倍。这些结果表明，FUra的掺入可损害催化性RNA分子的功能。
