High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking

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 nucleu s and, in addition, are concentrated in distinct nuclear substructures refe rred to as speckles. We have examined the intranuclear distribution and mob ility of the splicing factor U1 snRNP on a single-molecule level. Isolated U1 snRNPs were fluorescently labeled and incubated with digitonin-permeabil ized 3T3 cells in the presence of Xenopus egg extract. By confocal microsco py, U1 snRNPs were found to be imported into nuclei, yielding a speckled in tranuclear distribution. Employing a laser video-microscope optimized for h igh sensitivity and high speed, single U1 snRNPs were visualized and tracke d at a spatial precision of 35 nm and a time resolution of 30 ms. The singl e-particle data revealed that U1 snRNPs occurred in small clusters that col ocalized 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 opt ical axis, which corresponded to a mean effective diffusion coefficient of 1 mum(2)/s. An analysis of the trajectories of single U1 snRNPs revealed th at at least three kinetic classes of low, medium, and high mobility were pr esent. Moreover, the mean square displacements of these fractions were virt ually independent of time, suggesting arrays of binding sites. The results substantiate the view that nuclear speckles are not rigid structures but hi ghly dynamic domains characterized by a rapid turnover of U1 snRNPs and oth er splicing factors.