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

Institut für Physikalische und Theoretische Chemie, Rheinische Friedrich-Wilhelms-Universität, D-53115 Bonn, Germany.
Molecular Biology of the Cell (Impact Factor: 4.47). 01/2007; 17(12):5017-27. DOI: 10.1091/mbc.E06-06-0559
Source: PubMed


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 microm2/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.

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Available from: Ulrich Kubitscheck, Oct 04, 2015
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    • "Therefore, different strategies have been developed to discriminate between bound and free molecules in SMT (2,10,11). For example, bound molecules have been identified by setting two thresholds, an upper bound rmax for the maximum displacement of the molecule combined with a lower bound Nmin for the minimum number of time points comprising a valid bound-molecule track (10,23). "
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    • "As we have seen above that RNA tracking is possible, it would be interesting to also track single proteins as proteins are the facilitators of many major cellular processes. One such study looked at the mobility of fluorescently labeled uridine-rich small nuclear ribonucleoproteins (U1 snRNPs), biologically active splicing factors (Grunwald et al. 2006b). GFPlabeled ASF/SF2 was used to mark nuclear speckles allowing direct comparison of U1 snRNP dynamics inside and outside of the nuclear speckles. "
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