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

Abstract

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 digitoninpermeabilized 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 singleparticle 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 m 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.T he splicing of precursor mRNA in the nucleus is catalyzed by supramolecular assemblies designated as spliceosomes, which comprise more than 70 different proteins and five uridinerich small nuclear RNAs (snRNA; ref. 1). Most of these proteins and the snRNAs are organized in the Uridine-rich small nuclear ribonucleoproteins (U snRNPs), which are classified as U1, U2, U5, and U4͞U6, according to the snRNAs they contain. The snRNAs U1, U2, U4, and U5 are synthesized in the nucleus with a 5Ј-terminal monomethyl-guanosine (m 7 G)-cap structure, transiently exported into the cytoplasm, where a common set of seven core proteins (Sm proteins) bind to the snRNAs Sm site and form a ribonucleoprotein complex called ''Sm core'' (2). Stable association of all Sm proteins is necessary for hypermethylation of the m 7 G cap to the 2,2,7-trimethyl-guanosine (m 3 G)-cap structure (3, 4). Also, several proteins associate specifically with the individual U snRNPs; in the case of U1, those proteins are 70K, U1-A, and U1-C (5). After cap modification and 3Јend processing of the snRNAs (6), the mature snRNP particles are reimported into the nucleus by import receptors. The nuclear localization signal of U1 snRNPs is complex, with the m 3 G-cap structure representing one important signaling component (7,8). A second component is located at the Sm core but has not been defined precisely yet (9). Recent...

Enhancement of U4/U6 Small Nuclear Ribonucleoprotein Particle Association in Cajal Bodies Predicted by Mathematical Modeling

“…The scenario addressed is one in which a U4 snRNP moves by random walk throughout the nucleoplasm until it either associates with a U6 snRNP in the nucleoplasm or is captured by a CB, where it experiences a higher concentration of U6 snRNPs with which it can associate. The model assumes 1) that U4 and U6 snRNPs are targeted independently to CBs (Gerbi et al, 2003;Stanek et al, 2003;Stanek and Neugebauer, 2004), which was further verified by microinjection experiments presented here and (2) that snRNPs move within the nucleus by diffusion, as reported previously (Kues et al, 2001;Handwerger et al, 2003). Additional elements were empirically derived, including a three-dimensional model of the living HeLa cell nucleus and a determination of the average snRNP concentration difference (ϳ20-fold) in CBs versus nucleoplasm.…”

“…To validate the algorithm, the simulation was first used to compare the simulation results with the analytical solution solved for a simple sphere with a single target placed in its center (Kuthan, 2003). We considered a range of diffusion constants similar to those measured for RNPs of similar size and mobility to the U4 and U6 snRNPs: PAPB2 (D ϭ 0.6 m 2 /s), U7 snRNP (D ϭ 1.0 m 2 /s), and U1 snRNP (D ϭ 0.3 m 2 /s) (Kues et al, 2001;Calapez et al, 2002;Handwerger et al, 2003). Table 1 shows that two The Sm protein and U6 binding sites are indicated by black brackets.…”

“…Or, alternatively, is U4-U6 snRNP association by random walk throughout the nucleoplasm sufficiently rapid that concentration of snRNPs in CBs provides no advantage for snRNP assembly? We assume that the snRNPs move by diffusion within the nucleus, because the majority of previous studies have pointed to a lack of metabolic energy for intranuclear movements of proteins and RNAs (Misteli et al, 1997;Kues et al, 2001;Calapez et al, 2002;Dundr et al, 2004;Shav-Tal et al, 2004;Politz et al, 2006). To accomplish our aim, we determined the concentration difference for snRNAs in the CB versus nucleoplasm and derived a three-dimensional model of the living HeLa cell nucleus.…”

Dynamic tracking and mobility analysis of single GLUT4 storage vesicle in live 3T3-L1 cells

“…The lateral displacement of single vesicles could thus be obtained by estimating the change in position among consecutive images. This way we have successfully resolved the sub-pixel displacement of fluorescence labeled particles from consecutive images [18][19][20].…”

Single Molecule Fluorescence Monitoring in Eukaryotic Cells: Intranuclear Dynamics of Splicing Factors

“…This assumption was suggested by comparing the results of a previous study on intranuclear U1 snRNP mobility in our laboratory, which was performed not on living but rather on digitoninpermeabilized cells [28]. Digitonin-permeabilized cells are mostly physiologically inactive and splicing has ceased or is to a very large extent reduced.…”