Huang S, Mayeda A, Krainer A R, Spector D L
Cold Spring Harbor Laboratory, New York 11724, USA.
Mol Biol Cell. 1997 Jun;8(6):1143-57. doi: 10.1091/mbc.8.6.1143.
We have examined the effect of RCC1 function on the nuclear organization of pre-mRNA splicing factors and poly(A)+ RNA in the tsBN2 cells, a RCC1 temperature-sensitive mutant cell line. We have found that at 4-6 h after shifting cells from the permissive temperature (32.5 degrees C) to the restrictive temperature (39.5 degrees C), both small nuclear ribonucleoprotein particles and a general splicing factor SC35 reorganized into 4-10 large round clusters in the nucleus, as compared with the typical speckled distribution seen in cells at the permissive temperature. In situ hybridization to poly(A)+ RNA resulted in a similar pattern. Examination by double labeling demonstrated that the redistribution of splicing factors coincides with that of poly(A)+ RNA. Such changes in the nuclear organization of splicing factors and poly(A)+ RNA were not the result of the temperature shift or of chromatin condensation. Cellular transcription was not significantly altered in these cells and extracts made from both the permissive and restrictive temperature were splicing competent. Electron microscopic examination demonstrated that the large clusters containing both splicing factors and poly(A)+ RNA were fused interchromatin granule clusters. In addition, small electron-dense dot-like structures measuring approximately 80 nm in diameter were also observed, most of which are accumulated in enlarged interchromatin granule clusters in the nucleoplasm of RCC1- cells. In spite of the significant changes observed in the nucleoplasm, relatively little alteration was observed in nucleolar structure by both light and electron microscopic examination. The above observations suggest that the RCC1 protein directly or indirectly regulates the organization of splicing components and poly(A)+ RNA in the cell nucleus and that RCC1 may play a role in nuclear organization.
我们研究了RCC1功能对tsBN2细胞（一种RCC1温度敏感突变细胞系）中前体mRNA剪接因子和聚腺苷酸加尾RNA（poly(A)+ RNA）核组织的影响。我们发现，在将细胞从允许温度（32.5摄氏度）转移到限制温度（39.5摄氏度）后4至6小时，与在允许温度下细胞中典型的斑点状分布相比，小核核糖核蛋白颗粒和一般剪接因子SC35在细胞核中重新组织成4至10个大的圆形簇。对聚腺苷酸加尾RNA进行原位杂交得到了类似的模式。通过双重标记检查表明，剪接因子的重新分布与聚腺苷酸加尾RNA的重新分布一致。剪接因子和聚腺苷酸加尾RNA核组织的这种变化不是温度变化或染色质凝聚的结果。这些细胞中的细胞转录没有明显改变，并且从允许温度和限制温度制备的提取物都具有剪接活性。电子显微镜检查表明，包含剪接因子和聚腺苷酸加尾RNA的大簇是融合的染色质间颗粒簇。此外，还观察到直径约80纳米的小电子致密点状结构，其中大部分聚集在RCC1缺陷细胞的核质中扩大的染色质间颗粒簇中。尽管在核质中观察到了显著变化，但通过光学显微镜和电子显微镜检查，核仁结构的改变相对较小。上述观察结果表明，RCC1蛋白直接或间接调节细胞核中剪接成分和聚腺苷酸加尾RNA的组织，并且RCC1可能在核组织中发挥作用。
