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Vol. 17, Issue 3, 1239-1249, March 2006
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* Department of Biochemistry and Molecular Pharmacology and Program in Cell Dynamics, University of Massachusetts Medical School, Worcester, MA 01605;
Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01605;
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; and
German Cancer Research Center, 69120 Heidelberg, Germany
Submitted October 14, 2005;
Accepted December 2, 2005
Monitoring Editor: A. Gregory Matera
Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22°C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.
Abbreviations used: FCS, fluorescence correlation spectroscopy; mRFP-SC35, monomeric red fluorescent protein fused to SC35 protein; oligo, oligodeoxynucleotide.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: J.C.R. Politz (joan.politz{at}umassmed.edu).
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