UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute

Date

12-1-2006

Document Type

Article

Medical Subject Headings

Binding Sites; Cell Nucleus; Cell Survival; HeLa Cells; Humans; Kinetics; Protein Binding; Protein Transport; Ribonucleoprotein, U1 Small Nuclear

Abstract

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.

Comments

Citation: Grünwald D, Spottke B, Buschmann V, Kubitscheck U. Intranuclear binding kinetics and mobility of single native U1 snRNP particles in living cells. Mol Biol Cell. 2006 Dec;17(12):5017-27. doi:10.1091/mbc.E06-06-0559. Link to article on publisher's site

Copyright 2006 by The American Society for Cell Biology. Publisher PDF posted as allowed by the publisher's author rights policy at http://www.molbiolcell.org/site/misc/ifora.xhtml.

At the time of publication, David Grünwald was not yet affiliated with the University of Massachusetts Medical School.

Related Resources

Link to Article in PubMed

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.