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Vol. 17, Issue 12, 5017-5027, December 2006

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Intranuclear Binding Kinetics and Mobility of Single Native U1 snRNP Particles in Living Cells

David Grünwald^*,, Beatrice Spottke^*, Volker Buschmann^,, and Ulrich Kubitscheck^*

*Institut für Physikalische und Theoretische Chemie, Rheinische Friedrich-Wilhelms-Universität, D-53115 Bonn, Germany; and Max-Delbrück-Centrum, 13125 Berlin, Germany

Submitted June 27, 2006; Revised September 6, 2006; Accepted September 13, 2006
Monitoring Editor: Jennifer Lippincott-Schwartz

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay behavior on time scales ranging from milliseconds to seconds, reflecting the involvement of U1 snRNPs in numerous distinct interactions. The average dwell times for U1 snRNPs bound at sites within the nucleoplasm did not differ significantly from those in speckles, indicating that similar processes occur in both compartments. Mobile U1 snRNPs moved with diffusion constants in the range from 0.5 to 8 µm²/s. These values were consistent with uncomplexed U1 snRNPs diffusing at a viscosity of 5 cPoise and U1 snRNPs moving in a largely restricted manner, and U1 snRNPs contained in large supramolecular assemblies such as spliceosomes or supraspliceosomes.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Present addresses: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461;

PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany.

Address correspondence to: Ulrich Kubitscheck (u.kubitscheck{at}uni-bonn.de)

Abbreviations used: ASF, alternative splicing factor; EMCCD, electron multiplying CCD; FCS, fluorescence correlation spectroscopy; FRAP, fluorescence recovery after photobleaching; MSD, mean square displacements; SPT, single-particle tracking; snRNA, small nuclear RNA; MSD, mean square displacements; U snRNP, uridine-rich small nuclear ribonucleoprotein

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