Rapid, diffusional shuttling of poly(A) RNA between nuclear speckles and the nucleoplasm.
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 degrees 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.
Politz
Joan C Ritland
JC
Department of Biochemistry, University of Massachusetts Medical School, Worcester, MA 01605, USA. joan.politz@umassmed.edu
Tuft
Richard A
RA
Prasanth
Kannanganattu V
KV
Baudendistel
Nina
N
Fogarty
Kevin E
KE
Lifshitz
Larry M
LM
Langowski
Jörg
J
Spector
David L
DL
Pederson
Thoru
T
D000818:Animals; D022003:Cell Nucleus Structures / Q000378:metabolism*; D004058:Diffusion; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D034443:RNA Transport; D012333:RNA, Messenger / Q000378:metabolism* / Q000528:radiation effects; D051381:Rats; D012261:Ribonucleoproteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D000097573:Red Fluorescent Protein; C027903:oligo (dT); C073288:SRSF2 protein, human