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• W2153161471 abstract "Article2 August 1999free access Purification and biochemical characterization of interchromatin granule clusters Paul J Mintz Paul J Mintz Department of Molecular Genetics and Microbiology, S.U.N.Y. Stony Brook, Stony Brook, NY, 11794 USA Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Scott D Patterson Scott D Patterson Amgen Center, MS14-2-E, 1 Amgen Center Drive, Thousand Oaks, CA, 91320-1789 USA Search for more papers by this author Andrew F Neuwald Andrew F Neuwald Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Chris S Spahr Chris S Spahr Amgen Center, MS14-2-E, 1 Amgen Center Drive, Thousand Oaks, CA, 91320-1789 USA Search for more papers by this author David L Spector Corresponding Author David L Spector Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Paul J Mintz Paul J Mintz Department of Molecular Genetics and Microbiology, S.U.N.Y. Stony Brook, Stony Brook, NY, 11794 USA Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Scott D Patterson Scott D Patterson Amgen Center, MS14-2-E, 1 Amgen Center Drive, Thousand Oaks, CA, 91320-1789 USA Search for more papers by this author Andrew F Neuwald Andrew F Neuwald Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Chris S Spahr Chris S Spahr Amgen Center, MS14-2-E, 1 Amgen Center Drive, Thousand Oaks, CA, 91320-1789 USA Search for more papers by this author David L Spector Corresponding Author David L Spector Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Author Information Paul J Mintz1,2, Scott D Patterson3, Andrew F Neuwald2, Chris S Spahr3 and David L Spector 2 1Department of Molecular Genetics and Microbiology, S.U.N.Y. Stony Brook, Stony Brook, NY, 11794 USA 2Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY, 11724 USA 3Amgen Center, MS14-2-E, 1 Amgen Center Drive, Thousand Oaks, CA, 91320-1789 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:4308-4320https://doi.org/10.1093/emboj/18.15.4308 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Components of the pre-mRNA splicing machinery are localized in interchromatin granule clusters (IGCs) and perichromatin fibrils (PFs). Here we report the biochemical purification of IGCs. Approximately 75 enriched proteins were present in the IGC fraction. Protein identification employing a novel mass spectrometry strategy and peptide microsequencing identified 33 known proteins, many of which have been linked to pre-mRNA splicing, as well as numerous uncharacterized proteins. Thus far, three new protein constituents of the IGCs have been identified. One of these, a 137 kDa protein, has a striking sequence similarity over its entire length to UV-damaged DNA-binding protein, a protein associated with the hereditary disease xeroderma pigmentosum group E, and to the 160 kDa subunit of cleavage polyadenylation specificity factor. Overall, these results provide a key framework that will enable the biological functions associated with the IGCs to be elucidated. Introduction The mammalian cell nucleus is a highly organized structure in which DNA replication, transcription, pre-mRNA processing, ribosome biogenesis and RNA transport take place (for reviews, see Spector, 1993; Lamond and Earnshaw, 1998). The biochemical constituents and molecular mechanisms of these processes are reasonably well understood. However, less is known about how each of these processes is coordinated spatially and temporally within the structural framework of the nucleus. At present, only a few nuclear structures have been isolated by biochemical fractionation, i.e. nucleoli, nuclear pore–lamina complex and heterogeneous nuclear ribonucleoprotein particles (for a review, see Singer and Green, 1997). The isolation of these structures has facilitated the identification and functional characterization of their constituents, which enabled their biological roles to be substantiated. However, there are numerous other subnuclear structures including coiled bodies, the perinucleolar compartment (PNC), gems, cleavage bodies, promyelocytic leukemia (PML) bodies and interchromatin granule clusters (IGCs) which are less well characterized at the functional level because thus far it has not been possible to isolate them biochemically (for reviews, see Spector, 1993; Lamond and Earnshaw, 1998). Pre-mRNA splicing factors have been localized by immunofluorescence microscopy in a speckled nuclear distribution pattern which corresponds to perichromatin fibrils (PFs) and IGCs at the electron microscopic level (for a review, see Spector, 1993). PFs are fibrillar structures measuring 3–5 nm in diameter and are often found at the periphery of IGCs and distributed throughout the nucleoplasm (Monneron and Bernhard, 1969). In contrast to IGCs, PFs are labeled rapidly after short pulses with [3H]uridine, suggesting that they represent nascent transcripts (for a review, see Fakan, 1994). In support of this view, PFs are sensitive to RNase digestion and are reduced in cells treated with drugs that inhibit transcription by RNA polymerase II (Monneron and Bernhard, 1969; Petrov and Sekeris, 1971). IGCs, first identified by Swift (1959), represent the major part of the speckled staining pattern. IGCs measure 0.3–1.8 μm along their longest dimension (Thiry, 1995) and are composed of granules measuring 20–25 nm in diameter that appear to be connected in places by a 9–10 nm fiber (for reviews, see Fakan and Puvion, 1980; Spector, 1993). IGCs contain little to no DNA and are highly resistant to nuclease, detergent and high salt extractions (Monneron and Bernhard, 1969; Spector et al., 1983). Bromo-UTP and [3H]uridine incorporation studies have shown little to no labeling within IGCs, demonstrating that they are not centers of active transcription (for a review, see Spector, 1993). However, labeling has been found at the periphery of IGCs (Fakan and Nobis, 1978; Spector, 1990). Consistent with these findings, several studies have shown highly active genes and their transcripts to be associated with the periphery of speckled domains (for a review, see Huang and Spector, 1997). However, this association does not appear to be essential, as transcription can occur throughout the nucleoplasm (Zhang et al., 1994). Splicing factors have been shown to be recruited from IGCs to sites of active transcription (Jiménez-Garcıacute;a and Spector, 1993; Huang and Spector, 1996; Misteli et al., 1997). The molecular mechanism(s) for the recruitment of splicing factors to the sites of transcription is not completely understood. However, it has been shown that phosphorylation of the RS domain of SR proteins is involved in releasing these factors from IGCs and that dephosphorylation is at least part of the signal for the return of these factors to the IGCs (Misteli and Spector, 1996; Misteli et al., 1998). Recently, the C-terminal domain (CTD) of the largest subunit of RNA polymerase II has been shown to be involved in the intranuclear targeting of splicing factors to transcription sites in vivo (Misteli and Spector, 1999). Truncation of the CTD prevented the accumulation of splicing factors at a newly formed site of transcription, resulting in the inhibition of pre-mRNA splicing. While much has been learned about the nuclear distribution of IGCs (for reviews, see Fakan and Puvion, 1980; Spector, 1993; Thiry, 1995), the complete protein composition of these nuclear structures is not known. Elucidating the protein composition of IGCs is central to revealing the functions associated with this nuclear structure. We developed a biochemical nuclear fractionation protocol that enabled us to separate IGCs from chromatin, nuclear lamins, nucleoli and other nuclear bodies. We employed a novel mass spectrometry strategy and peptide microsequencing to identify rapidly many of the 75 proteins that are enriched in this nuclear structure. These data provide a key framework that will enable the biological functions associated with the IGCs to be elucidated. Results Biochemical purification and morphological characterization of IGCs Adult mouse liver nuclei contain 20–30 irregularly shaped IGCs each measuring ∼0.3–1.8 μm along their longest axis (Figure 1A). In order to elucidate the biochemical composition of IGCs, we have developed a cell fractionation procedure to isolate IGCs from mouse liver nuclei (Figure 1B). Nuclei were isolated by sucrose gradient sedimentation (Berezney and Coffey, 1977). This procedure produced clean nuclei with little to no cytoplasmic contamination (Figure 2A). Figure 1.(A) Transmission electron micrograph of mouse liver nucleus. Thin section of a nucleus showing interchromatin granule clusters (IGCs) (arrowheads). Bar: 1 μm. (B) Flow chart of the biochemical purification of IGCs. Download figure Download PowerPoint Figure 2.Morphological characterization of the IGC fractionation. All samples were post-stained using the EDTA-regressive method which preferentially stains ribonucleoproteins (Bernhard, 1969). (A) The nuclear pellet contains well preserved nuclei. The IGCs, nucleoli (N), nuclear lamina (L) and chromatin (C) are readily observed. (B) Thin section of the nuclear pellet after 1% Triton X-100, DNase I and 0.5 M NaCl treatments. The IGCs are still well preserved, whereas the nucleoli appear partially extracted. Little to no chromatin is present in the extracted nuclei. (C) The cesium sulfate pellet contains nuclear lamina (L), nucleoli (N) and residual nuclear components (asterisk). Bars: 2 μm. Download figure Download PowerPoint In order to purify IGCs away from other nuclear structures, we proceeded first to remove the nuclear membrane by using a non-ionic detergent, 1% Triton X-100 (data not shown). Next, DNA was digested with DNase I and chromatin was released by several sequential extractions with 0.5 M NaCl. After the nuclease digestion and salt extractions, three major types of nuclear structures still remained visible: nuclear lamina, nucleoli and IGCs (Figure 2B). The IGCs remained well preserved whereas the nucleoli appeared to have been extracted after the 0.5 M NaCl treatments. In order to separate the IGCs from other nuclear structures and release them from the nuclear framework, the nuclei were disrupted mechanically in the presence of 5 mM dithiothreitol (DTT) and the resultant homogenate was layered onto a 0.25 M cesium sulfate solution followed by low-speed centrifugation. The cesium sulfate pellet contained predominantly nuclear lamina, nucleoli and residual nuclear components (Figure 2C). The IGCs, which were enriched in the cesium sulfate supernatant, were retrieved by high-speed centrifugation (Figure 3A and B). Figure 3A shows a low magnification electron micrograph of a thin section of the IGC pellet revealing a relatively homogenous population of IGCs and little to no apparent contamination by other nuclear structures. The isolated interchromatin granules measured between 18 and 27 nm in diameter (Figure 3B), similar to their in situ counterparts. To confirm that the purified IGCs contained splicing factors, thin sections were immunolabeled with 3C5 monoclonal antibody (Turner and Franchi, 1987), which recognizes a family of SR proteins, followed by colloidal gold-conjugated secondary antibody. The colloidal gold particles labeled the IGCs (Figure 3C), whereas the control sections, which were not incubated with primary antibody, showed little to no labeling (Figure 3D). These data demonstrate, at a structural level, the high degree of purity of the IGC fraction. Figure 3.Transmission electron micrographs of the purified IGC fraction. (A) Low magnification view of the purified IGCs showing a homogenous population Bar: 0.5 μm. (B) High magnification view of the IGCs showing intact and well-preserved interchromatin granules. Differences in staining intensity reflect the plane of section. Bar: 200 nm. (C) The IGC fraction was labeled with monoclonal antibody 3C5, which recognizes phosphorylated SR proteins, followed by 5 nm colloidal gold-conjugated secondary antibody Bar: 200 nm; inset: 50 nm. (D) Control section labeled only with the secondary antibody shows little to no immunogold labeling. Bar: 200 nm. Download figure Download PowerPoint Biochemical composition of IGCs To investigate the protein composition of each step in the IGC purification, we performed SDS–PAGE using 4–20% gradient gels. Such analyses showed a complex protein composition in all of the fractions examined (Figure 4A). Different proteins were enriched in various fractions. For example, a group of proteins in the 10–15 kDa range were enriched in the Triton pellet, several proteins in the 60–70 kDa range were enriched in the cesium sulfate pellet, and a group of three proteins around 220 kDa were enriched in the IGC pellet. Previous studies have used immunopurification to obtain highly specific macromolecular complexes (e.g. hnRNPs) or organelles (Kvalheim et al., 1987; Piñol-Roma et al., 1988; Saucan and Palade, 1994). In order to address whether the protein composition of the purified IGC pellet fraction is specific, and not due to other proteins randomly co-sedimenting with the IGCs, we have purified IGCs further by using magnetic beads coupled with a splicing factor antibody (3C5). The immunopurified IGCs showed a protein composition similar to that of the isolated IGC fraction when examined by SDS–PAGE (Figure 4A and B, lanes 5 and 1, respectively). As shown in the control lanes (Figure 4B, lanes 2 and 3), there was little to no binding of the IGCs to beads that did not contain coupled primary antibody. Since the protein composition of the immunopurified fraction was similar to that of the initial IGC pellet fraction, all further analyses were performed on the IGC pellet fraction. Figure 4.One- and two-dimensional gel electrophoresis. (A) All pellet fractions from the IGC purification were separated on a 4–20% SDS gradient gel. Equal amounts (40 μg) of protein were loaded. (B) Magnetic beads coupled with 3C5 monoclonal antibody were used to purify the IGCs further. Immunopurified IGCs (lane 1) show a protein composition similar to that of the biochemically purified IGCs. Control beads (lanes 2 and 3) show no non-specific proteins. (C and D) The IGC or nuclear pellets were first separated by 2.7% polyacrylamide using pH 3.5–10 ampholites in the first dimension followed by 12.5% SDS–PAGE in the second dimension. The proteins were visualized by silver staining. (C) Nuclear pellet (100 μg). (D) IGC pellet (100 μg). Approximately 75 protein spots were enriched in the IGC fraction as indicated by the circled regions. Download figure Download PowerPoint In order to identify at greater resolution the constituents of the IGCs, purified IGCs were analyzed by two-dimensional gel electrophoresis. Approximately 300 protein spots were identified in the IGC fraction (Figure 4D), 75 of which were enriched when compared with the nuclear fraction (Figure 4C). The degree of enrichment for each spot varied. For example, a series of three spots just above the 20 kDa marker were almost undetectable in the two-dimensional gel of whole nuclei and were significantly enriched in the IGC fraction (Figure 4C and D). Two additional spots, just below the 10 kDa marker, were present in the nuclear fraction and were significantly enriched in the IGCs. Since IGCs are known to contain pre-mRNA splicing factors, we were interested next in determining their presence in the purified IGC fraction as a means of further assessing its purity. This was accomplished by immunoblotting the purified IGC fraction. Several pre-mRNA splicing and non-splicing factors including SF3a66, SF2/ASF, U2AF65 and U2AF35 were enriched in the IGC fraction (Figure 5A, top row). We found that several other pre-mRNA processing-related proteins, U1-70K, U2-B″ and PAB II, while present in the IGC fraction, were not enriched (Figure 5A, bottom row). This may be due to their more loose association with the IGCs. Interestingly, the hyperphosphorylated form of the largest subunit of RNA polymerase II is also present in the IGC fraction, but is not enriched. This result was not unexpected since it has been shown that this form of the polymerase is present in nuclear speckles (Bregman et al., 1995). Several other nuclear proteins that have been shown by immunofluorescence to be localized to other nuclear compartments or to be distributed diffusely throughout the nucleoplasm, such as hnRNP A1, lamins A and C, ribosomal protein S6 and the PML protein show little to no immunoreactivity in the IGC fraction (Figure 5B). It must be pointed out here that it is not possible to quantitate the fold enrichment for the IGCs accurately since thus far all of the proteins that have been localized to this structure are also present diffusely throughout the nucleoplasm. The diffuse nuclear pool represents factors that are at transcription sites as well as those that are in transit between IGCs and transcription sites. Figure 5.Immunoblot analysis of the IGC fraction. (A) Immunoblots in the upper row show several pre-mRNA splicing factors that are enriched in the IGC fraction. The lower row shows several other splicing factors and RNA polymerase II to be present, but not enriched, in the IGC fraction. (B) Immunoblots using antibodies that recognize proteins associated with other cellular compartments show little to no immunoreactivity in the IGC fraction. Equal amounts of protein (10 μg) were loaded. Download figure Download PowerPoint To understand better where in our fractionation scheme proteins associated with different nuclear compartments are enriched preferentially, we immunoblotted all of the pellet fractions from each step in the IGC purification. Figure 6A shows that lamin B1 is enriched predominantly in the cesium sulfate pellet, consistent with our observations of this fraction by transmission electron microscopy (Figure 2C). Interestingly, nucleolar protein B23 (nucleophosmin) (Ochs et al., 1983) was not enriched in the IGC or cesium sulfate pellets (Figure 6B, lanes 5 and 4, respectively). Most of nucleolar protein B23 was removed by the three 0.5 M NaCl extractions (Figure 6B, lane 3). Previous studies have shown that some hnRNPs are also extractable from nuclei at 0.5 M NaCl (Beyer et al., 1977; Piñol-Roma et al., 1988). We examined the distribution of hnRNP C1/C2 proteins across our fractionation and found that most of these proteins were removed by the nuclease digestion and salt extractions (Figure 6C, lane 3). We have also examined the distribution of SR proteins and found that most of the SR proteins were enriched in the IGC fraction when compared with the nuclear pellet (Figure 6D, lanes 5 and 1, respectively). In addition, there was little to no immunoreactivity in the cesium sulfate pellet. These data, along with the SDS–PAGE analysis, strongly support the view that the IGCs were biochemically purified and demonstrate the complex protein composition of IGCs. Figure 6.Further characterization of purified IGCs. Immunoblots were probed with antibodies against (A) lamin B1, (B) nucleolar protein B23, (C) hnRNP C1/C2 or (D) 3C5 antibody to a family of SR proteins. Proteins associated with different nuclear structures were enriched in different fractions. Download figure Download PowerPoint Mass spectrometry analysis In recent years, analysis by mass spectrometry has been a powerful approach used to identify the protein components of large macromolecular complexes rapidly by subjecting individual gel bands to analysis (for reviews, see Lamond and Mann, 1997; Patterson, 1998). For example, several recent reports have used this approach to identify the protein constituents of in vitro assembled spliceosomes (Neubauer et al., 1998), yeast U1 small nuclear ribonucleoprotein particles (Neubauer et al., 1997) and the yeast spindle pole (Wigge et al., 1998). Here we have used a novel mass spectrometry approach to identify many of the IGC proteins rapidly. The entire IGC protein mixture was digested enzymatically and subjected to liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) in a data-dependent manner followed by uninterpreted fragment ion searching of non-redundant and expressed sequence tag databases (dbEST). This is the first time that a large and complex protein mixture has been digested and subjected to mass spectrometry analysis en toto. Purified IGCs (denatured, reduced and alkylated) were first subjected to partial trypsin digestion. The complex peptide mixture was fractionated by HPLC with on-line ion-trap electrospray mass spectrometry. In two separate runs, we have thus far identified 33 known proteins (Table I) and ESTs encoding at most 16 proteins (to date) after searching a non-redundant protein database or dbEST (NCBI and DDBJ/EMBL/GenBank) with the uninterpreted MS/MS spectra. A group of 19 proteins (identified from 71 spectra), previously shown to be localized to IGCs, is composed largely of splicing factors. One member of this group, a previously characterized RNA/DNA-binding protein (RNPS1) has been identified recently as SF7A, a general activator of pre-mRNA splicing (A.R.Krainer and A.Mayeda, personal communication). In addition to splicing factors, two proteins related to different types of cancer were identified. A peptide sequence corresponding to a region of the Skip protein was identified. Skip has been shown to be associated with the Ski oncoprotein (Dahl et al., 1998). The Skip protein was also identified when spliceosomes were analyzed by mass spectrometry (Neubauer et al., 1998) and, in addition, it was shown to be localized to nuclear speckles. A second protein, TLS-associated serine-arginine (TASR) protein (Yang et al., 1998), which is associated with the TLS (translocated in liposarcoma) protein, was also identified in the IGC fraction. In addition, our analysis identified a subset of proteins (14 identified from 22 MS/MS spectra) that have been characterized in a different biological context and thus far have not been shown to be present in IGCs (Table I). Some of these proteins (e.g. histones and ribosomal proteins) may be contaminants based upon their previous functional associations. However, we cannot rule out the possibility that they may also be minor constituents of the IGCs. Finally, we have identified several new proteins (Table I) and ESTs (data not shown) associated with the IGCs. Table 1. Proteins identified in purified IGC fraction Peptide sequence No. of peptides identified No. of times identified Accession No. Previously characterized proteins Splicing factor SF2/ASF IYVGNLPPDIR 5 7 q07955 Splicing factor SC35 ELRVQMAR 2 3 q01130 Splicing factor SRp20 VYVGNLGNNGNKTELER 4 7 x91656 Splicing factor SRp55 ALDKLDGTEINGR 1 2 u30829 Splicing factor SRp75 LIVENLSSR 3 5 q08170 U5 snRNP 116 kDa protein IAVEPVNPSELPK 1 1 u97079 snRNP Sm D2 protein NNTQVLINCR 1 1 p43330 snRNP Sm D3 protein VAQLEQVYIR 1 1 p43331 snRNP-associated protein RVLGLVLLR 4 4 p27048 RNA/DNA-binding protein (RNPS1) GYAYVEFENPDEAEK 1 1 s43417 hnRNP A2/B1 QEMQEVQSSR 1 1 p22626 hnRNP A3 SSGSPYGGGYGSGGGSGGYGSR 1 2 p51991 hnRNP M MGPAMGPALGAGIER 10 17 p52272 hnRNP G DYAPPPRDYTYR 4 6 p38159 hnRNP D IFVGGLSPDTPEEK 1 1 d55671 hnRNP E1 IANPVEGSSGR 1 2 s42472 Nuclear protein Skip YTPSQQGVAFNSG 1 2 q13573 TLS-ass. with SR repeats (TASR) YLRPPNTSLFVR 3 5 af042383 Heat shock protein 70 TTPSYVAFTDTER 3 3 p08109 Lamin A SGAQASSTPLSPTR 1 1 p48678 Lamin B1 ASAPATPLSPTR 1 1 p14733 Lamins C1/C2 SLETENAGLR 3 3 p11516 nhp2/rs6 family protein LLDLVQQSCNYK 3 3 p55770 CBF5 VAKLDTSQWPLLLK 1 1 u59151 Histone H2a AGLQFPVGR 3 6 s04152 Histone H2b AMGIMNSFVNDIFER 3 6 p10853 Histone H3 DIQLAR 1 2 p06351 Histone H4 DNIQGITKPAIR 3 4 s03427 Importin β-1 subunit VLANPGNSQVAR 1 1 p70168 Ribosomal protein S11 YYKNISSR 1 1 c76740 60S ribosomal protein L7a AGVNTVTTLVENK 1 1 p12970 60S ribosomal protein L6 AVDLQILPK 1 1 p47911 60S ribosomal protein L18 ILTFDQLALESPK 1 1 p35980 New IGC proteins Plenty-of-proline (POP)a AVTIATPATAAPAAVSAATTTSA… 1 3 w13917 KIAA0324a SLLPNSSQDELMEVEK 2 4 ab002322 KIAA0017 a,b KFVIHPESNNLIIIETD 2 2 d13642 a Co-localization of YFP fusion proteins with an endogenous splicing factor in vivo. b Protein identified by peptide microsequencing. The identified peptide sequences were used to screen protein databases. Two peptide sequences SLLPNSSQDELMEVEK and LGLIQEDVASSCIPR (matched to Amgen EST sequences) were used to identify a full-length human cDNA clone, KIAA0324, after searching a non-redundant protein database (DDBJ/EMBL/GenBank accession No. AB002322). The open reading frame (ORF) of KIAA0324 contains many RS/SR dipeptide repeats and a putative nuclear localization signal (data not shown). We did not find any potential homologs of KIAA0324 in the databases. The predicted molecular mass of this protein is ∼125.6 kDa and it is a highly basic protein (pI = 12). To verify that this protein is a bona fide constituent of the IGCs, a fusion protein was made with yellow fluorescent protein (YFP) and expressed in HeLa cells by transient transfection (Figure 7A). The transfected cells were also immunostained with anti-SC35 monoclonal antibody which diagnostically identifies nuclear speckles (Figure 7B). The expressed fusion protein co-localizes with the endogenous SC35 splicing factor (Figure 7C). In addition, the fusion protein was also present in the cytoplasm, suggesting that it may shuttle. Figure 7.Characterization of KIAA0324 and KIAA0017. The human full-length KIAA0324 (A–C) or KIAA0017 (D–F) cDNAs were fused to yellow fluorescent protein (YFP). HeLa (A–C) or BHK (D–F) cells were transiently transfected and immunostained with the anti-SC35 (B) or anti-B″ (E) antibodies. The fusion proteins localized in nuclear speckles and diffusely in the cytoplasm. (C) and (F) are the respective merged images of (A) and (B) and (D) and (E). Bars: 10 μm; 5 μm. Download figure Download PowerPoint A third peptide (AVTIATPATAAPAAVSAATTTSAQEEPAAAPEPR) was matched to a full-length mouse cDNA clone called Plenty-of-Proline (POP) (DDBJ/EMBL/GenBank accession No. AF062655). The POP protein is highly basic (pI = 11.9) and has a predicated molecular mass of ∼101 kDa. The ORF of POP contains many RS/SR dipeptide repeats, several proline-rich regions and a putative NLS (data not shown). A homolog to POP, SRm160, was identified (DDBJ/EMBL/GenBank accession No. AF048977), which is a human splicing co-activator previously localized to speckles (Blencowe et al., 1998). Using a sequence alignment program (NCBI, BLAST 2 Sequences), POP shares 90% identity and 92% similarity with SRm160 (data not shown). We have also made a POP–YFP fusion protein and have confirmed that POP is a bona fide component of the IGCs (data not shown). The remaining 16 ESTs currently do not match to any identified full-length cDNAs. Microsequence analysis of the 140 kDa protein In addition to mass spectrometry, peptide microsequencing of gel bands was used to identify new proteins of the IGC fraction. An enriched 140 kDa gel band of the IGC fraction was excised from a Coomassie-stained SDS–polyacrylamide gradient gel for microsequencing. The peptide sequences obtained (KFVIHPESNNLIIIETD and KNVSEELDRTPPEVSK) were used to search a non-redundant protein database, and a full-length cDNA clone, KIAA0017, was identified (DDBJ/EMBL/GenBank accession No. D13642). The protein is acidic (pI = 5.1) and has a predicted molecular mass of ∼136.6 kDa. The ORF of KIAA0017 contains a putative NLS (data not shown); however, no other known motifs were identified. Database searching using the KIAA0017 sequence has allowed us to identify closely related proteins in other metazoans (Figure 8) (Caenorhabditis elegans and Drosophila melanogaster), in a plant (Arabidopsis thaliana) and in a protozoan (Plasmodium falciparum), suggesting that human protein KIAA0017 performs an important, perhaps essential, biological function that arose early during eukaryotic evolution. Given their high degree of sequence identity to KIAA0017 (43–61%), these proteins are likely to perform the same function in these diverse organisms as KIAA0017 does in humans. Notably, even budding yeast, which lacks IGCs, encodes a member of this family. The function of this yeast protein may be somewhat distinct from that of these other proteins, however, given its lower degree of sequence identity to KIAA0017 (25% identity). Figure 8.Alignment of protein sequences related to the human hypothetical protein KIAA0017. The alignment was generated using PSI-BLAST (Altschul et al., 1997) and conserved residues were highlighted using an automated procedure (Neuwald et al., 1999); minor adjustments in the alignment were made to improve readability. Protein designations are color coded by family using the following scheme: KIAA0017, red; UV-DDB, blue; CPSF 160 kDa subunit, black. H" @default.
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• W2153161471 title "Purification and biochemical characterization of interchromatin granule clusters" @default.
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