Wang W, Krug R M
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1179, USA.
RNA. 1998 Jan;4(1):55-64.
The influenza virus NS1 protein inhibits the splicing of the major class of mammalian pre-mRNAs (GU-AG Introns) by binding to a specific stem-bulge in U6 snRNA, thereby blocking the formation of U4/U6 and U2/U6 complexes. The splicing of the minor class of AT-AC introns takes place on spliceosomes that do not contain U6 snRNA, but rather U6atac snRNA-a highly divergent U6 snRNA counterpart. Nonetheless, we demonstrate that the NS1 protein inhibits AT-AC splicing in vitro, and specifically binds to only U6atac snRNA among the five minor class snRNAs. Chemical modification/interference assays show that the NS1 protein binds to the stem-bulge near the 3' end of U6atac snRNA, encompassing nt 82-95 and nt 105-114. Although the sequence of this stem-bulge differs significantly from the sequence of the stem-bulge to which the NS1 protein binds in U6 snRNA, RNA competition experiments Indicate that U6 and U6atac snRNAs likely share the same binding site on the NS1 protein. Previously, the region of U6atac snRNA containing this 3' stem-bulge had not been implicated in any interactions of this snRNA with either U4atac or U12 snRNA. However, as assayed by psoralen crosslinking, we show that the NS1 protein inhibits the formation of U12/U6atac complexes, but not the formation of U4atac/U6atac complexes. We can conclude that the inhibition of AT-AC splicing results largely from the inhibition of formation of U12/U6atac complexes caused by the binding of the NS1 protein to the 3' stem-bulge of U6atac snRNA.
流感病毒NS1蛋白通过与U6 snRNA中的特定茎环结构结合，抑制哺乳动物主要类型前体mRNA（GU-AG内含子）的剪接，从而阻止U4/U6和U2/U6复合物的形成。少数类型的AT-AC内含子的剪接发生在不含U6 snRNA而是含有U6atac snRNA（一种高度分化的U6 snRNA对应物）的剪接体上。尽管如此，我们证明NS1蛋白在体外抑制AT-AC剪接，并且在五种少数类型的snRNA中仅特异性结合U6atac snRNA。化学修饰/干扰实验表明，NS1蛋白结合在U6atac snRNA 3'端附近的茎环结构上，包括第82 - 95位核苷酸和第105 - 114位核苷酸。虽然该茎环结构的序列与NS1蛋白在U6 snRNA中结合的茎环结构序列有显著差异，但RNA竞争实验表明U6和U6atac snRNAs可能在NS1蛋白上共享相同的结合位点。此前，含有该3'茎环结构的U6atac snRNA区域未涉及该snRNA与U4atac或U12 snRNA的任何相互作用。然而，通过补骨脂素交联分析，我们表明NS1蛋白抑制U12/U6atac复合物的形成，但不抑制U4atac/U6atac复合物的形成。我们可以得出结论，AT-AC剪接的抑制主要是由于NS1蛋白与U6atac snRNA的3'茎环结构结合导致U12/U6atac复合物形成受到抑制。
