Reduced Mobility of the Alternate Splicing Factor (Asf) through the Nucleoplasm and Steady State Speckle Compartments

Abstract

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.

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

“…These findings are consistent with numerous studies on the mobilities of nuclear proteins that have demonstrated rapid exchange between the nucleoplasm and speckles (Kruhlak et al, 2000;Phair and Misteli, 2000;Molenaar et al, 2004), the nucleolus (Phair and Misteli, 2000;Snaar et al, 2000;Chen and Huang, 2001), and Cajal bodies (Snaar et al, 2000;Handwerger et al, 2003;Dundr et al, 2004). We also show here that the SC35 protein is extremely dynamic in both within and outside speckles.…”

“…Our results do not rule out the presence of a very small slowmoving or immobile class of poly(A) RNA in the nucleus, but if it exists, our results indicate that it is Ͻ5% of the total poly(A) RNA and is present in similar amounts in both speckles and the nucleoplasm. Therefore, it is unlikely that poly(A) RNA serves as an immobile scaffolding to form the speckle; rather, it can move freely in and out of speckles in all directions and visit other speckles.These findings are consistent with numerous studies on the mobilities of nuclear proteins that have demonstrated rapid exchange between the nucleoplasm and speckles (Kruhlak et al, 2000;Phair and Misteli, 2000;Molenaar et al, 2004), the nucleolus (Phair and Misteli, 2000;Snaar et al, 2000;Chen and Huang, 2001), and Cajal bodies (Snaar et al, 2000;Handwerger et al, 2003;Dundr et al, 2004). We also show here that the SC35 protein is extremely dynamic in both within and outside speckles.…”

“…A similar phenomenon was observed in live cell tracking of 28S rRNA, which was observed to move between nucleoli (Politz et al, 2003). Certain nuclear proteins have also been observed to move between nuclear compartments, such as fibrillarin between nucleoli and SF2/ASF between speckles (Kruhlak et al, 2000;Phair and Misteli, 2000), but these proteins are not destined for transport to the cytoplasm, as is the aforementioned 28S rRNA and polyadenylated mRNA. It therefore seems that even macromolecules that must be transported to the cytoplasm can roam freely not only throughout the nucleoplasm but also enter and exit similar nuclear compartments with no direct requirement of metabolic energy.…”

Tissue-specific Expression and Dynamic Organization of SR Splicing Factors inArabidopsis

“…However, after blocking transcription, we found that almost all of the above-mentioned dynamic events ceased, indicating that these dynamic events are also related to transcriptional activities in the plant nuclei. Similar dynamic events were also observed in mammalian cell nuclei (Misteli et al, 1997;Kruhlak et al, 2000;Phair and Misteli, 2000).…”

“…However, after blocking transcription, we found that almost all of the above-mentioned dynamic events ceased, indicating that these dynamic events are also related to transcriptional activities in the plant nuclei. Similar dynamic events were also observed in mammalian cell nuclei (Misteli et al, 1997;Kruhlak et al, 2000;Phair and Misteli, 2000).It is well documented that splicing of most pre-mRNAs occurs cotranscriptionally in mammalian cells (for review, see Proudfoot et al, 2002). As transcription inhibition does not disassemble the speckles in plant nuclei, and the distribution of splicing factors in highly metabolic cells such as meristematic cells is more diffuse, we propose that the plant speckles are storage or modification/assembly sites of splicing components, from which splicing factors or preassembled spliceosome subcomplexes are recruited to transcription sites similar to that suggested for their mammalian counterparts (for review, see Lamond and Spector, 2003).…”