SMALL NUCLEAR RIBONUCLEOPROTEIN (RNP)-U2 CONTAINS NUMEROUS ADDITIONAL PROTEINS AND HAS A BIPARTITE RNP STRUCTURE UNDER SPLICING CONDITIONS
SMALL NUCLEAR RIBONUCLEOPROTEIN (RNP)-U2 CONTAINS NUMEROUS ADDITIONAL PROTEINS AND HAS A BIPARTITE RNP STRUCTURE UNDER SPLICING CONDITIONS
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DOI：
10.1128/mcb.13.1.307
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发表时间：
1993-01-01
影响因子：
5.3
通讯作者：
LUHRMANN, R
中科院分区：
文献类型：
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作者：
BEHRENS, SE;TYC, K;LUHRMANN, R
小核（sn）核糖核蛋白（RNP）U2通过识别未剪接的前体mRNA的分支位点而在mRNA的剪接中起作用。当HeLa核剪接提取物在甘油梯度上离心时，U2 snRNP在12S（在高盐浓度条件下）或17S（在低盐浓度条件下）处沉淀。我们在非变性条件下通过离心和免疫亲和层析从剪接提取物中分离17S U2 snRNP，并通过电子显微镜检查其结构。除了存在于12 S U2 snRNP中的常见蛋白质B '、B、D1、D2、D3、E、F和G以及U2特异性蛋白质A'和B“之外，至少9种先前未鉴定的表观分子量为35、53、60、66、92、110、120、150和160 kDa的蛋白质与17 S U2 snRNP结合。后一种蛋白质在高于200 mM的盐浓度下与U2 snRNP解离，产生12S U2 snRNP颗粒。在电子显微镜下，17S U2 snRNP表现出二分的外观，两个主要的球状结构域连接的短丝状结构，是敏感的RNA酶。这些发现表明，12S U2 snRNP中不存在的额外球状结构域包含一些17S U2特异性蛋白。当U2 snRNP为17S形式时，U2 snRNP中RNA的5'端比其为12S形式时更暴露于与RNA酶H和化学探针的反应。去除该RNA的5'末端将snRNP的Svedberg值从17S降低至12S。沿着17 S snRNP的特殊形态，这些数据表明大多数17 S U2特异性蛋白质与U2 snRNA的5'端结合。
Small nuclear (sn) ribonucleoprotein (RNP) U2 functions in the splicing of mRNA by recognizing the branch site of the unspliced pre-mRNA. When HeLa nuclear splicing extracts are centrifuged on glycerol gradients, U2 snRNPs sediment at either 12S (under high salt concentration conditions) or 17S (under low salt concentration conditions). We isolated the 17S U2 snRNPs from splicing extracts under nondenaturing conditions by using centrifugation and immunoaffinity chromatography and examined their structure by electron microscope. In addition to common proteins B', B, D1, D2, D3, E, F, and G and U2-specific proteins A' and B'', which are present in the 12S U2 snRNP, at least nine previously unidentified proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa bound to the 17S U2 snRNP. The latter proteins dissociate from the U2 snRNP at salt concentrations above 200 mM, yielding the 12S U2 snRNP particle. Under the electron microscope, the 17S U2 snRNPs exhibited a bipartite appearance, with two main globular domains connected by a short filamentous structure that is sensitive to RNase. These findings suggest that the additional globular domain, which is absent from 12S U2 snRNPs, contains some of the 17S U2-specific proteins. The 5' end of the RNA in the U2 snRNP is more exposed for reaction with RNase H and with chemical probes when the U2 snRNP is in the 17S form than when it is in the 12S form. Removal of the 5' end of this RNA reduces the snRNP's Svedberg value from 17S to 12S. Along with the peculiar morphology of the 17S snRNP, these data indicate that most of the 17S U2-specific proteins are bound to the 5' half of the U2 snRNA.