Kues T, Dickmanns A, Lührmann R, Peters R, Kubitscheck U
Institut für Medizinische Physik und Biophysik, Westfälische Wilhelms-Universität, Robert-Koch-Strasse 31, D-48149 Münster, Germany.
Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):12021-6. doi: 10.1073/pnas.211250098. Epub 2001 Oct 2.
Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are components of the splicing machinery that removes introns from precursor mRNA. Like other splicing factors, U snRNPs are diffusely distributed throughout the nucleus and, in addition, are concentrated in distinct nuclear substructures referred to as speckles. We have examined the intranuclear distribution and mobility of the splicing factor U1 snRNP on a single-molecule level. Isolated U1 snRNPs were fluorescently labeled and incubated with digitonin-permeabilized 3T3 cells in the presence of Xenopus egg extract. By confocal microscopy, U1 snRNPs were found to be imported into nuclei, yielding a speckled intranuclear distribution. Employing a laser video-microscope optimized for high sensitivity and high speed, single U1 snRNPs were visualized and tracked at a spatial precision of 35 nm and a time resolution of 30 ms. The single-particle data revealed that U1 snRNPs occurred in small clusters that colocalized with speckles. In the clusters, U1 snRNPs resided for a mean decay time of 84 ms before leaving the optical slice in the direction of the optical axis, which corresponded to a mean effective diffusion coefficient of 1 microm(2)/s. An analysis of the trajectories of single U1 snRNPs revealed that at least three kinetic classes of low, medium, and high mobility were present. Moreover, the mean square displacements of these fractions were virtually independent of time, suggesting arrays of binding sites. The results substantiate the view that nuclear speckles are not rigid structures but highly dynamic domains characterized by a rapid turnover of U1 snRNPs and other splicing factors.
富含尿苷的小核核糖核蛋白（U snRNP）是从前体mRNA中去除内含子的剪接机制的组成部分。与其他剪接因子一样，U snRNP在细胞核中呈弥散分布，此外，还集中在被称为斑点的不同核亚结构中。我们在单分子水平上研究了剪接因子U1 snRNP在细胞核内的分布和移动性。将分离的U1 snRNP进行荧光标记，并在非洲爪蟾卵提取物存在的情况下与经洋地黄皂苷通透处理的3T3细胞一起孵育。通过共聚焦显微镜观察发现，U1 snRNP被导入细胞核，呈现出斑点状的核内分布。使用针对高灵敏度和高速进行优化的激光视频显微镜，以35 nm的空间精度和30 ms的时间分辨率对单个U1 snRNP进行可视化和跟踪。单粒子数据显示，U1 snRNP以小簇的形式出现，与斑点共定位。在这些簇中，U1 snRNP在沿光轴方向离开光学切片之前平均停留衰减时间为84 ms，这对应于平均有效扩散系数为1 微米²/秒。对单个U1 snRNP轨迹的分析表明，至少存在低、中、高三种不同移动性的动力学类别。此外，这些组分的平均平方位移实际上与时间无关，表明存在结合位点阵列。这些结果证实了核斑点不是刚性结构，而是以U1 snRNP和其他剪接因子的快速周转为特征的高度动态区域的观点。
