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 fac tor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move i n 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) usin g time-lapse microscopy and the technique fluorescence recovery after photo bleaching (FRAP). We find that ASF-GFP moves at rates up to 100 times slowe r than free diffusion when it is associated with speckles and, surprisingly , also when it is dispersed in the nucleoplasm. The mobility of ASF is cons istent with frequent but transient interactions with relatively immobile nu clear 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 im mobile binding sites.