Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments.
Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF-GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF-GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.
Kruhlak
M J
MJ
Department of Anatomy, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
Lever
M A
MA
Fischle
W
W
Verdin
E
E
Bazett-Jones
D P
DP
Hendzel
M J
MJ
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002451:Cell Compartmentation / Q000502:physiology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D004058:Diffusion; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D018668:Muntjacs; D009687:Nuclear Proteins / Q000378:metabolism*; D010777:Photochemistry; D047428:Protein Kinase Inhibitors; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D014157:Transcription Factors / Q000037:antagonists & inhibitors; D014158:Transcription, Genetic / Q000187:drug effects
D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D000068103:Serine-Arginine Splicing Factors; D012319:RNA Polymerase II