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Abstract：
The small nuclear RNAs U4 and U6 display extensive sequence complementarity and co-exist in a single ribonucleoprotein particle. We have investigated intermolecular base-pairing between both RNAs by psoralen cross-linking, with emphasis on the native U4/U6 ribonucleoprotein complex. A mixture of small nuclear ribonucleoproteins U1 to U6 from HeLa cells, purified under non-denaturing conditions by immune affinity chromatography with antibodies specific for the trimethylguanosine cap of the small nuclear RNAs was treated with aminomethyltrioxsalen. A psoralen cross-linked U4/U6 RNA complex could be detected in denaturing polyacrylamide gels. Following digestion of the cross-linked U4/U6 RNA complex with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate cross-linked fragments. These fragments were analysed by chemical sequencing methods and their positions identified within RNAs U4 and U6. Two overlapping fragments of U4 RNA, spanning positions 52 to 65, were cross-linked to one fragment of U6 RNA (positions 51 to 59). These fragments show complementarity over a contiguous stretch of eight nucleotides. From these results, we conclude that in the native U4/U6 ribonucleoprotein particle, both RNAs are base-paired via these complementary regions. The small nuclear RNAs U4 and U6 became cross-linked in the deproteinized U4/U6 RNA complex also, provided that small nuclear ribonucleoproteins were phenolized at 0 degree C. When the phenolization was performed at 65 degrees C, no cross-linking could be detected upon reincubation of the dissociated RNAs at lower temperature. These results indicate that proteins are not required to stabilize the mutual interactions between both RNAs, once they exist. They further suggest, however, that proteins may well be needed for exposing the complementary RNA regions for proper intermolecular base-pairing in the course of the assembly of the U4/U6 RNP complex from isolated RNAs. Our results are discussed also in terms of the different secondary structures that the small nuclear RNAs U4 and U6 may adopt in the U4/U6 ribonucleoprotein particle as opposed to the isolated RNAs.
摘要：
小核RNAU4和U6显示出广泛的序列互补性,并且在单个核糖核蛋白颗粒中共存。我们已经通过补骨脂素交联研究了两种RNA之间的分子间碱基配对，重点是天然U4/U6核糖核蛋白复合物。来自HeLa细胞的小核核糖核蛋白U1至U6的混合物，在非变性条件下通过免疫亲和层析纯化，用氨甲基三氧沙林处理小核RNA的三甲基鸟苷帽特异性抗体。可以在变性聚丙烯酰胺凝胶中检测到补骨脂素交联的U4/U6RNA复合物。用核糖核酸酶T1消化交联的U4/U6RNA复合物后，使用变性聚丙烯酰胺凝胶中的二维对角电泳分离交联片段。通过化学测序方法分析这些片段,并鉴定它们在RNAU4和U6中的位置。U4RNA的两个重叠片段，跨越位置52至65,与U6RNA的一个片段(位置51至59)交联。这些片段在8个核苷酸的连续片段上显示互补性。从这些结果来看,我们得出结论，在天然U4/U6核糖核蛋白颗粒中，这两种RNA通过这些互补区域进行碱基配对。小核RNAU4和U6也在去蛋白化的U4/U6RNA复合物中交联，前提是小的核核糖核蛋白在0℃下进行酚化。当酚化在65℃下进行时，在较低温度下重新孵育解离的RNA时，无法检测到交联。这些结果表明，不需要蛋白质来稳定两种RNA之间的相互作用，一旦他们存在。他们进一步建议，然而,在从分离的RNA组装U4/U6RNP复合物的过程中，可能需要蛋白质来暴露互补RNA区域以进行适当的分子间碱基配对。我们的结果还根据小核RNAU4和U6可能在U4/U6核糖核蛋白颗粒中采用的不同二级结构进行了讨论，而不是分离的RNA。
