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1. Enhanced proteolysis of pre-mRNA splicing factors in myeloid cells.
Shav-Tal Y, Lee B, Bar-Haim S, Vandekerckhove J, Zipori D. Exp Hematol. 2000, 28:1029-38 OBJECTIVE: Molecular identification and characterization of the bone marrow nuclear protein detected by the B92 monoclonal antibody. MATERIALS AND METHODS: The protein was purified to homogeneity from acute myeloid leukemia cells and was subjected to peptide digestion and amino acid sequencing. Identified sequences were used to screen a bone marrow cDNA library in search of matching transcripts. The protein was further studied in different cells and tissues by examination of protease inhibitors and harsh lytic conditions and during apoptosis in HL-60 cells. RESULTS: We found that the apparent bone marrow specific protein is a 47 kD proteolytic cleavage product of PSF, an essential pre-mRNA splicing factor. PSF is completely cleaved to p47 during lysis of immature myeloid cells due to potent proteolytic activity found in these cells but is rare in other cells and tissues. Furthermore, p47 is abundant in intact normal and tumor myeloid cells while in other cell types it is undetectable. The cleavage of PSF is accompanied by digestion of the PTB splicing regulator but not other proteins tested. In contrast, during apoptosis PTB is degraded while PSF remains intact. CONCLUSIONS: The bone marrow 47 kD protein is a fragment constituting the N-terminal, protease-resistant half of the splicing factor PSF. Proteolytic degradation of PSF specifically occurs in intact myeloid cells and this process is enhanced upon myeloid cell lysis.
Shav-Tal Y, Cohen M, Lapter S, Dye B, Patton JG, Vandekerckhove J, Zipori D.
Mol Biol Cell. 2001, 12:2328-40
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.
3. Reorganization of nuclear factors during myeloid differentiation.
Shav-Tal Y, Lee BC, Bar-Haim S, Schori H, Zipori D.
J Cell Biochem 2001, 81:379-92
Differentiation in several stem cell systems is associated with major morphological changes in global nuclear shape. We studied the fate of inner-nuclear structures, splicing factor-rich foci and Cajal (coiled) bodies in differentiating hemopoietic, testis and skin tissues. Using antibodies to the splicing factors PSF, U2AF(65) and snRNPs we find that these proteins localize in foci throughout the nuclei of immature bone marrow cells. Yet, when granulocytic cells differentiate and their nuclei condense and become segmented, the staining localizes in a unique compact and thread-like structure. The splicing factor-rich foci concentrate in the interior of these nuclei while the nuclear periphery and areas of highly compact chromatin remain devoid of these molecules. Differentiated myeloid cells do not stain for p80 coilin, the marker for Cajal bodies. Immature myeloid cells contain Cajal bodies although these usually do not coloclaize with PSF-rich foci. Following complete inhibition of transcription in myeloid cells, the threaded PSF pattern becomes localized in several foci in the different lobes of mature granulocytes while in human HL-60 immature myeloid leukemia cells PSF is found in the perinucleolar compartment. Studies of other differentiating stem cell systems show that PSF staining disappears completely in differentiated, transcriptionally inactive sperm cells, is scarce as cells migrate from the inner skin layers outward and is lost as cells of the hair follicle mature. We conclude that the formation and distribution of splicing factor-rich foci in the nucleus during differentiation of various cell lineages is dependent on the levels of chromatin condensation and the differentiation status of the cell.
Shav-Tal Y, Blechman J, Darzacq X, Montagna C, Dye BT, Patton JG, Singer RH, Zipori D.
Mol Biol Cell. 2005, 5:2395-413
Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.
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