EVIDENCE FOR THE EXISTENCE OF SNRNAS U4 AND U6 IN A SINGLE RIBONUCLEOPROTEIN COMPLEX AND FOR THEIR ASSOCIATION BY INTERMOLECULAR BASE-PAIRING
EVIDENCE FOR THE EXISTENCE OF SNRNAS U4 AND U6 IN A SINGLE RIBONUCLEOPROTEIN COMPLEX AND FOR THEIR ASSOCIATION BY INTERMOLECULAR BASE-PAIRING
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DOI：
10.1002/j.1460-2075.1984.tb01977.x
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发表时间：
1984-01-01
期刊：
影响因子：
11.4
通讯作者：
LUHRMANN, R
中科院分区：
文献类型：
--
作者：
BRINGMANN, P;APPEL, B;LUHRMANN, R
来自真核细胞[埃利希细胞，HeLa细胞]的小的核核糖核蛋白颗粒（snRNP）可以在用对2，2，7-三甲基鸟苷（m3 G）具有高亲和力的抗体制备的亲和柱上分级，所述m3 G存在于snRNA的5“-末端帽中。虽然snRNP U1、U2和U 5在0.1 M盐存在下用核苷m3 G洗脱，但snRNP物质U4和U6仅在盐浓度增加时解吸。对于来自HeLa或埃利希腹水肿瘤细胞的snRNP同样观察到相同的分级模式。由于U6 RNA缺乏m3 G残基，并且其RNA不与抗m3 G反应，因此其与U4 RNP在抗m3 G亲和柱上的共层析表明，离散的snRNP U4和U6在核提取物中密切相关，或者两种RNA都组织在1个RNP颗粒中。通过在蔗糖梯度中使用纯化的snRNP进行沉降研究，获得了U4/U6 RNP颗粒的进一步证据。RNA的凝胶分级显示snRNA U4和U6在梯度中的相同分布，并且U4/U6 RNP颗粒比snRNP U1或U2沉积得更快。沉降期间snRNP U4和U6之间的物理缔合通过它们与来自梯度级分的抗m3 G IgG的共沉淀来显示。提供了snRNA U4和U6通过U4/U6 RNP颗粒中的分子间碱基配对而结合的实验证据，如通过以下发现所证明的：在0 ℃下对U4/U6 RNP进行酚化后，抗m3 G IgG使U6 RNA与U4 RNA共沉淀。C.当在65 ℃下进行酚化时，C 2 RNA解离，抗m3 G IgG仅沉淀U4 RNA。在颗粒沉降研究中，发现U 5 RNP是沉降最慢的snRNP物质，这表明它与其他RNP之一不相关，因此作为离散的RNP颗粒存在。
Small nuclear ribonucleoprotein particles (snRNP) from eukaryotic cells [Ehrlich cells, HeLa cells] can be fractionated on affinity columns prepared with antibodies of high affinity for 2,2,7-trimethylguanosine (m3G), which is present in the 5''-terminal caps of the snRNA. While the snRNP U1, U2 and U5 are eluted with the nucleoside m3G in the presence of 0.1 M salt, the snRNP species U4 and U6 are only desorbed when the salt concentration is increased. The same fractionation pattern was likewise observed for snRNP from HeLa or Ehrlich ascites tumor cells. Since U6 RNA lacks the m3G residue and its RNA does not react with anti-m3G, its co-chromatography with U4 RNP on anti-m3G affinity columns suggests either that discrete snRNP U4 and U6 are intimately associated in nuclear extracts or that both RNA are organized in 1 RNP particle. Further evidence for a U4/U6 RNP particle is obtained by sedimentation studies with purified snRNP in sucrose gradients. Gel fractionation of RNA shows identical distributions of snRNA U4 and U6 in the gradient, and the U4/U6 RNP particle sediments faster than the snRNP U1 or U2. Physical association between snRNP U4 and U6 during sedimentation is shown by their co-precipitation with anti-m3G IgG from the gradient fractions. Experimental evidence is provided that snRNA U4 and U6 are associated by intermolecular base pairing in the U4/U6 RNP particle, as demonstrated by the finding that anti-m3G IgG co-precipitates U6 RNA with U4 RNA following phenolization of U4/U6 RNP at 0.degree. C. When the phenolization is performed at 65.degree. C the 2 RNA dissociate and anti-m3G IgG solely precipitates U4 RNA. In the particle sedimentation studies U5 RNP was found to be the slowest sedimenting snRNP species, which indicates that it is not associated with one of the other RNP and therefore exists as a discrete RNP particle.