Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein Interactions

Abstract

The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1-70K and SR proteins and therefore may acquire new functions. INTRODUCTIONSplicing factors are found within the nucleus both in a diffuse form and in distinct domains termed interchromatin granules (IG) or "speckles" (Spector, 1993). These functional compartments are spatially dynamic with regard to movement and composition (Eils et al., 2000). Splicing factors within IGs are highly mobile and are constantly associating and dissociating from their compartments (Phair and Misteli, 2000). The mAb, B92, which recognizes the polypyrimidine tract binding protein (PTB)-associated splicing factor (PSF), produces typical specked nuclear pattern in immunostaining. These PSF speckles disappear during granulocytic differentiation (Lee et al., 1996;Shav-Tal et al., 2000). We recently showed that this disappearance is associated with partial degradation (Shav-Tal et al., 2000) and by the formation of alternative nuclear structures (Shav-Tal et al., 2001). The present study indicates that PSF speckles disappear also through a different mechanism, involving conformational changes that cause breakdown of PSF speckles and relocalization of the molecule in the nucleus. The functional nature of speckles is a matter of some controversy (for review, see Park and De Boni, 1999). These structures have been suggested to play a role in storage and recycling of splicing factors (Jimenez-Garcia and Spector, 1993), whereas a portion may act as reservoirs for the recruitment to active sites of transcription (Huang and Spector, 1996;Zeng et al., 1997). This view does not predict a direct involvement of speckles in pre-mRNA splicing. On the other hand, it has been shown that nascent mRNA transcripts and transcription sites are closely associated with speckles, that transcription occurs at the surface of speckles (Clemson and Lawrence, 1996), and that microinjected pre-mRNAs have high affinity to speckles (Wang et al., 1991). It has thus been proposed that speckles can act coordinately in transcription and splicing and that pre-mRNA splicing can take place bo...

Dynamic Sorting of Nuclear Components into Distinct Nucleolar Caps during Transcriptional Inhibition

“…After longer exposure to the drug these compartments took upon a more rounded structure. We have previously shown that GFP-PSF in apoptotic cells, induced by prolonged exposure to ActD, formed large, rounded structures (Shav-Tal et al, 2001a). This correlated with hyperphosphorylation of PSF in apoptotic and mitotic cells.…”

Binding of mouse VL30 retrotransposon RNA to PSF protein induces genes repressed by PSF: Effects on steroidogenesis and oncogenesis

“…The PSF protein sequence is tightly conserved between mice and humans (21), suggesting that a regulatory mechanism involving human PSF and a human homolog of mVL30 also could be conserved. We have identified complete retrotransposon sequences in the human genome that are Ͼ90% identical to the mVL30 sequence (unpublished results), suggesting that human VL30 RNA could have regulatory roles analogous to those of mVL30 RNA in mice.…”

whitesnake/sfpq is required for cell survival and neuronal development in the zebrafish

“…PSF was first identified as an essential pre-mRNA splicing factor (Patton et al, 1993) and has since been shown to exhibit multiple functions in nucleic acid synthesis and processing in vitro and in tissue culture, including transcriptional corepression, DNA unwinding, and linking RNA transcripts with RNA polymerase II (Emili et al, 2002;Shav-Tal and Zipori, 2002). In addition, it has been suggested to play a role in tumorigenesis as well as apoptosis, as sfpq translocation occurs in papillary renal cell carcinoma (Clark et al, 1997), and nuclear relocaliza-tion and hyperphosphorylation of PSF occur during apoptosis (Shav-Tal et al, 2001).…”