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Vol. 15, Issue 8, 3876-3890, August 2004
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* Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724;
Amgen Center, Thousand Oaks, California 91320-1789
Submitted March 25, 2004;
Accepted May 20, 2004
Monitoring Editor: Joseph Gall
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Lamond and Earnshaw, 1998; Misteli, 2000). Although some nuclear functions can be reproduced in in vitro systems (i.e., transcription and pre-mRNA splicing), these systems may be less efficient than their in vivo counterparts (Corden and Patturajan, 1997). Therefore, in vivo spatial and temporal coordination may have a significant influence on gene expression and other nuclear processes. Among those nuclear organelles thus far identified in normal and cancer cells (for a review, see Spector, 2001) are interchromatin granule clusters (IGCs), perichromatin fibrils, nucleoli, paraspeckles, perinucleolar compartment, Cajal bodies, gemini of Cajal bodies, and promyelocytic leukemia nuclear bodies. Several of these organelles have been shown to have a relationship to various disease states, including cancer and spinal muscular atrophy (Spector et al., 1992; Matera, 1999; Huang, 2000). Recently, several nuclear structures, including the nuclear pore complex (Rout et al., 2000; Cronshaw et al., 2002), nuclear envelope (Schirmer et al., 2003), and nucleoli (Andersen et al., 2002; Scherl et al., 2002) have been isolated, and their protein composition was characterized by mass spectrometry analysis. In addition, in vitro-assembled spliceosomes, the U1 small nuclear ribonucleoprotein particle (snRNP), and the U4/U6.U5 tri-snRNP have been analyzed using this approach (Neubauer et al., 1997, 1998; Gottschalk et al., 1999; Rappsilber et al., 2002; Zhou et al., 2002). Analysis of the yeast nuclear pore complex (NPC) identified 174 proteins in total of which 40 were found to be associated with the NPC in the form of nucleoporins (29 proteins) or transport factors (11 proteins) (Rout et al., 2000). In the case of the NPC from rat liver nuclei, 94 proteins in total were identified, 29 of which were classified as nucleoporins and 18 were classified as NPC-associated proteins (Cronshaw et al., 2002). By using a subtractive proteomics approach to analyze a mouse nuclear envelope fraction, 13 known nuclear envelope integral proteins were identified as well as 67 uncharacterized open reading frames with predicted membrane spanning regions (Schirmer et al., 2003). Proteomic analysis of human nucleoli has identified 271 (Andersen et al., 2002) to 
350 (Scherl et al., 2002) proteins, 30% of which are encoded by novel human genes (Andersen et al., 2002). Analysis of in vitro assembled spliceosomes has identified 145 (Zhou et al., 2002) or 311 proteins (Rappsilber et al., 2002).
One of the most intensely studied nuclear substructures, the IGCs, are thought to play a role in efficiently coupling transcription and pre-mRNA splicing in nuclei (for a review, see Lamond and Spector, 2003). IGCs measure 1.0–1.5 µm along their widest length and are composed of clusters of 20- to 25-nm granules that often seem to be connected by short fibers (for a review, see Fakan and Puvion, 1980). The IGCs were initially shown to contain a subset of pre-mRNA splicing factors by immunofluorescence and immunoelectron microscopy (for a review, see Spector, 1993). More recent studies have shown that the IGCs are enriched in a number of pre-mRNA splicing factors and the large subunit of RNA polymerase II (Bregman et al., 1995; Mortillaro et al., 1996), however, transcription and pre-mRNA splicing do not generally seem to take place within these nuclear regions (Cmarko et al., 1999; Misteli and Spector, 1999). Instead, splicing factor assembly, modification and/or storage are thought to occur within these nuclear compartments (for a review, see Misteli and Spector, 1998; Lamond and Spector, 2003). IGCs are dynamic nuclear structures from which splicing factors have been shown to be recruited to sites of active transcription in living cells (Misteli et al., 1997; Janicki et al., 2004). Studies using fluorescence recovery after photobleaching have shown that there is a continuous flux of proteins between the IGCs and the nucleoplasm (Kruhlak et al., 2000; Phair and Misteli, 2000). However, it is unclear whether the IGC proteins move as monomers, small complexes, or as a large complex such as individual 20- to 25-nm granules to sites of transcription. In addition, the specific composition of individual interchromatin granules remains to be determined.
We have previously established a protocol to biochemically isolate IGCs from mouse liver nuclei (Mintz et al., 1999) and in our initial characterization of this fraction by mass spectrometry, we identified 33 protein constituents of IGCs. Here, we have extended these studies to saturation and have identified 146 IGC proteins as well as 32 novel protein candidates. We have characterized the 146 proteins based upon their motifs and localization. Our analysis has identified 31 RS domain-containing proteins as well as proteins involved in other aspects of mRNA metabolism. Interestingly, we have found a significant overlap (63%) between our analysis and the recently reported analyses of the protein composition of spliceosomes (Neubauer et al., 1998; Rappsilber et al., 2002; Zhou et al., 2002). Our findings support a proposed role of IGCs in the assembly, modification, and/or storage of proteins involved in pre-mRNA processing.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The purified IGC fraction was directly dissolved in 2 M urea-phosphate-buffered saline-0.1 mM EDTA, allowing us to recover IGC proteins with high efficiency, rather than our previous approach, whereby we resuspended proteins in TM5 (0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 5 mM MgCl2). In addition, in the present study we started with 6 times the number of mice relative to our previous report, which yielded 10 times more IGC proteins based on measurement of protein concentrations by mass spectrometry analysis. One-third of the dissolved IGC proteins were biotinylated at Cys residues with the chemical cross-linker Biotin-HPDP followed by trypsin digestion, whereas the remaining two-thirds of the IGC proteins were directly digested with trypsin. Cys-containing peptides were selected through avidin chromatography to reduce the complexity of the peptide mixture, thus increasing the chances of detecting low abundant peptides with Cys residues that are normally masked by abundant peptides (Spahr et al., 2000). The selected Cys-containing peptides, as well as a mixture of trypsin-digested peptides without Cys selection, were analyzed by liquid chromatography and tandem mass spectrometry (MS/MS). Fragment ion spectra were batch searched against nonredundant protein sequences in databases. Resulting peptide matches were manually evaluated and confirmed. Motif analysis of each identified protein was performed using SMART (http://smart.embl-heidelberg.de/) (Schultz et al., 1998; Letunic et al., 2002). Database for Tables 1, 2, 3, 4 is available at http://spectorlab.cshl.edu.
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Transient Transfection of Cells and Immunofluorescence Microscopy
Four cDNA clones that correspond to newly identified IGC proteins (KIAA0164, 0670, 0801, and 1019) were kindly provided by Dr. Nagase (Kazusa DNA Research Institute, Chiba, Japan). The clones were fused in frame, to enhanced yellow fluorescent protein at their N termini by using the pEYFP-C expression vector (BD Biosciences Clontech, Palo Alto, CA). A431 cells were transfected with the resultant constructs using FuGENE6 transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions and incubated for 16 h. Cells were processed for immunofluorescence as described previously (Spector et al., 1998). Antibody to SC35 (Fu and Maniatis, 1990) was used at 1:1000 dilution to label IGCs, followed by Texas Red-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Images were acquired on an Axioplan 2i fluorescence microscope (Carl Zeiss, Thornwood, NY)with a plan-APO 100x/1.4 numerical aperture objective lens using Openlab Software (Improvision, Lexington, MA) and an Orca charge-coupled device camera (Hammamatsu, Middlesex, NJ).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and expressed sequence tags (ESTs) encoding at most 16 proteins after searching a nonredundant protein database or dbEST (National Center for Biotechnology Information and DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank) with the uninterpreted MS/MS spectra. We have now extended this study by scaling up our purification and optimizing the sample preparation (see MATERIALS AND METHODS) to identify a larger complement of IGC proteins. The IGC fraction was digested with trypsin and subjected to liquid chromatography electrospray ionization MS/MS followed by uninterpreted fragment ion searching of nonredundant and expressed sequence tag databases (dbEST) in a data-dependent manner. Our analysis will identify proteins that are enriched in IGCs and therefore localize in a speckled pattern by immunofluorescence microscopy (i.e., snRNPs and serine-arginine proteins), as well as other proteins that may be equally distributed throughout the nucleoplasm, including the IGCs and diffuse nuclear pools (i.e., hnRNP A and C). We performed five rounds of the analysis and reached saturation as we repeatedly obtained the same set of peptide sequences. As a result, 2214 peptide sequences were obtained, which correspond to 360 proteins. We categorized the proteins based upon their known function, motifs, and/or localization: identified IGC proteins (41%), potential IGC proteins (19%), novel IGC protein candidates (9%), and unexpected IGC proteins (31%) (Tables 1, 2, 3, 4).
Identified IGC Proteins
The group of identified IGC proteins (Table 1, 146 proteins) contains the most frequently detected proteins and is composed of previously identified IGC proteins, as well as proteins whose functions are similar to well-characterized IGC proteins. Because many of the proteins that have been localized to IGCs contain RNA binding motifs and RS domains that are stretches of dipeptide repeats of arginine (R) and serine (S) (Birney et al., 1993), we systematically surveyed all of the detected proteins with regard to these motifs. Nineteen percent of the identified IGC proteins contain an RS domain, and 50% contain one to four RNA binding motifs (Table 1). The presence of an RS domain and/or basic region has been reported to act as a speckle localization signal for some pre-mRNA splicing factors as well as a protein interaction domain (for a review, see Fu, 1995; Graveley, 2000). In addition, each of the identified proteins was characterized with regard to the presence of other motifs and its localization to nuclear speckles. Twenty-seven percent of the identified IGC proteins have previously been reported to localize in nuclear speckles. We did not detect any sequence motifs common to all identified IGC proteins. Two frequently detected motifs in this group are the DEAD box helicase motif (Linder et al., 1989; Luking et al., 1998) and an RNA binding motif (Birney et al., 1993). The absence of a specific localization signal, aside from the RS domain contained within a subset of proteins, may reflect a more transient interaction of many proteins with nuclear speckles or may indicate that these proteins are targeted to and/or associate with nuclear speckles through other RS-domain–containing interaction partners.
A profile of this protein group (Figure 1) indicates that 54% of the identified IGC proteins have a role in pre-mRNA splicing, 20% of the proteins are classified as RNA-associated proteins, and 7% have roles in other aspects of pre-mRNA processing, such as 3' RNA processing, mRNA export, and nonsense-mediated decay (see DISCUSSION). Together, 81% of the IGC proteins likely participate in pre-mRNA/mRNA metabolism.
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IGCs have been proposed to be important for the coupling of RNA polymerase II transcription and pre-mRNA splicing, because numerous proteins are recruited from nuclear speckles to sites of transcription (for a review, see Lamond and Spector, 2003). Six percent of the identified IGC proteins are involved in transcription (Table 1). Several subunits of RNA polymerase II, including the largest subunit, which has previously been localized to nuclear speckles (Bregman et al., 1995; Mortillaro et al., 1996), and several transcription factors have been identified in this fraction. Most general transcription factors were diffusely distributed throughout the nucleoplasm and were not identified in the IGC fraction. However, the proportion of transcription factors may be underrepresented, because we have categorized many proteins as potential IGC proteins (Table 2) due to the lack of information on their specific subnuclear localization. As expected, we did not detect RNA polymerases I or III in the IGC fraction.
Interestingly, several proteins were identified that have previously been characterized as having structural roles in cells. These proteins include actin (Nakayasu and Ueda, 1984), matrin 3 (Belgrader et al., 1991; Nakayasu and Berezney, 1991), lamin A/C (Jagatheesan et al., 1999), and pinin (Ouyang and Sugrue, 1996; Brandner et al., 1997; Ouyang et al., 1997). Although all of these proteins have been localized to IGCs, they do not form an underlying protein scaffold for attachment of IGCs (Sacco-Bubulya and Spector, 2002). Instead, they may be integral components of individual interchromatin granules and their role(s) is yet to be determined.
In addition, our analysis identified several proteins that were recently shown by others to have roles in pre-mRNA splicing or to be localized to nuclear speckles. These include acinus (Boucher et al., 2001; Schwerk et al., 2003), eIF4Aiii (Li et al., 1999; Holzmann et al., 2000), RNA binding motif protein 8 (Y14) (Kataoka et al., 2001), and the RNA export protein Aly (Zhou et al., 2000). Surprisingly, our analysis did not reveal some proteins that have previously been reported to localize to nuclear speckles, for example, casein kinase II and protein phosphatase 1 (Trinkle-Mulcahy et al., 2001). Protein phosphatase 1 has only one trypsin cleavage site, so it would likely be underrepresented in our peptide identification by mass spectrometry. Other proteins that were not identified associate with IGCs with low affinity and therefore may dissociate during the purification procedure. Alternatively, association of proteins such as kinases and phosphatases may be more sensitive to changes in phosphorylation state during IGCs purification.
Potential and Unexpected IGC Proteins
We found 70 proteins whose nuclear localizations, for the most part, have not been characterized, although these proteins have been studied at the biochemical and/or molecular levels (Table 2). We categorized this group of proteins as potential IGC proteins. Many of these potential IGC proteins have roles in transcription, such as DNA cis-element binding factors (i.e., transcription factor NF-AT45, nuclear factor I-X, and C/EBPs), components of a chromatin remodeling complex (BAF53A and BAF57), and transcription mediators (transcriptional coactivator CRSP77, thyroid hormone receptor-associated proteins, and transcriptional intermediary factors). Seven percent of the potential IGC proteins are possible molecular chaperones because they contain either a cyclophilin type peptidyl-prolyl cis-trans-isomerase motif or AAA ATPase family motif. Four percent are DNA repair proteins, and the remaining proteins have varied functions or they have not been studied at the molecular level. Although the subnuclear distribution of each protein remains to be determined, the identification of these proteins in the IGC fraction suggests that IGCs may be major sites for coupling transcription and pre-mRNA processing, thus promoting efficient gene expression. Furthermore, some of the molecular chaperone proteins included in this category may be responsible for the formation/maintenance of the structure of IGCs.
To determine whether these proteins are bona fide IGC constituents, we made cDNA fusion constructs to tag them with yellow fluorescent protein and expressed them in A431 cells. Four representative cDNAs shown in Figure 2 all encoded for proteins that localize to IGCs (KIAA0670, KIAA0801, and KIAA1019) or their periphery (KIAA0164), further confirming the specificity of our preparation. Because we now have evidence that they are bona fide IGC proteins, we have included these four proteins in Table 1.
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In our previous study, we showed that the IGC fraction was highly purified and free of detectable contaminants, such as other nuclear structures. When examined using transmission electron microscopy, the final fraction was significantly homogeneous, containing granules measuring 20–25 nm in diameter that were immunolabeled with anti-SC35 antibody, a marker protein for IGCs (Mintz et al., 1999). In addition, immunoblot analysis showed that a subset of known IGC proteins are highly enriched in the IGC fraction, whereas minimal contamination of protein components of other nuclear structures, such as the nuclear envelope, promyelocytic leukemia bodies, or Cajal bodies were detected in the IGC fraction (Mintz et al., 1999). Nonetheless, by mass spectrometry we did detect numerous proteins, which have previously been characterized as components of other cellular structures, and therefore we have classified them as unexpected proteins (Table 3). Because these proteins are relatively abundant and mass spectrometry is a highly sensitive technique, it is likely that they are protein contaminants in our preparation.
Novel IGC Protein Candidates
In addition, and most interestingly, we found 32 proteins for which no available biological information is available, except for sequence information (Table 4). Each of these proteins was analyzed for known motifs. Four proteins have various similarities to other proteins involved in RNA metabolism. These examples include a protein with a RNA helicase C-terminal domain (KIAA0052), a protein slightly similar to cleavage and polyadenylation stimulation factor (KIAA0663), a putative splicing factor (RIKEN cDNA 2410002M20), and a protein with similarity to SAF-B (similar to KIAA0138 gene product), which is known to be in IGCs. Thus, these proteins are highly likely to be IGC components. Two other proteins contain an SAP motif, one also with a poly-A binding domain (RIKEN cDNA 2610511G16) and the other with SPRY and Ffh domains (similar to hypothetical protein). The SAP motif is named after SAF-A/B, acinus and PIAS (Aravind and Koonin, 2000). SAF-B and acinus are localized in the IGCs (Table 1 and Figure 2), and PIAS has been shown to be associated with RNA helicase II/ATP-dependent RNA helicase (Valdez et al., 1997). The SAP motif is defined as a sequence homologous to the N-terminal DNA binding region of SAF-A and has been found in several other nuclear proteins (Aravind and Koonin, 2000). Proteins with a SAP domain often contain an additional motif that is involved in the assembly of RNA-processing complexes (Aravind and Koonin, 2000). Therefore, it has been proposed that such proteins are associated both with chromatin and RNA. Additionally, they may function to deliver the RNA processing machinery to the site of transcription (Aravind and Koonin, 2000), which overlaps with a proposed function of IGCs.
RS Domain-containing Proteins
In the IGC proteomic analysis, we detected 31 proteins with RS dipeptide motifs, including two novel IGC candidates (Tables 1, 2, and 4). Of these, 17 proteins have actually been shown to localize to IGCs by either immunofluorescence analysis or expression of the fluorescently tagged proteins in cells (Table 1). By comparing these proteins, based upon the organization of their other motifs relative to the RS domains, we sorted them into three major groups (Figure 3). The first group (Figure 3A) represents proteins with an RS motif and one to three RNA recognition motifs (RRMs). This group can be further divided into three subgroups. Proteins in the first subgroup, from SRp20 to SRp75, are small proteins with N-terminal RRMs and a C-terminal RS motif. Among this group are members of the SR family of pre-mRNA splicing factors (SRp20, SF2/ASF, SC35, 9G8, SRp30, SRp40, SRp55, and SRp75). Proteins in the second sub-group, from the tra-2 beta homologue to splicing factor HCC1, are also small splicing factors, but they have an N-terminal RS motif and a C-terminal RRM(s). Proteins in the third subgroup are related to the first two subgroups because they have N-terminal RRM and C-terminal or middle region RS motifs; however, they are larger proteins and their RS motifs are continuous to RD or RE dipeptides, which could provide them with additional functional properties (see DISCUSSION).
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Proteins in the second group (Figure 3B) are medium-to-large proteins, ranging from 663 to 2297 amino acids. All (except for acinus) do not have a recognizable RRM motif, and they are characterized by the presence of compositionally biased regions. Among them, Btf and a protein called "similar to TRAP150" have significant sequence similarities to TRAP150 (60 and 33% sequence identity, respectively). TRAP150 has been shown to be a transcriptional mediator component (Johnson et al., 2002). Proteins categorized in this second group contain additional domains, such as a cyclophilin type peptidyl-prolyl cis-trans-isomerase (proisomerase) domain, a SAP domain, and a DEAD box helicase motif, thus they may have additional interactions and/or functions. Indeed, SRm 300 is a splicing coactivator (Blencowe et al., 2000), and acinus is involved in chromatin condensation in the late stage of apoptosis (Sahara et al., 1999) as well as in pre-mRNA processing (Schwerk et al., 2003). Btf also was reported to be involved in apoptosis (Kasof et al., 1999).
The third group (Figure 3C) also represents proteins of medium-to-large (917–2427 amino acid length) size with interesting repetitive sequences. Especially notable is son protein, which contains six types of repetitive sequences that cover approximately one-third of its sequence. The functions of these proteins are not well characterized; however, NP220 was reported to be a DNA and nuclear matrix binding protein (Inagaki et al., 1996), and SR140 is associated with U2 snRNP (Will et al., 2002).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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IGCs and Spliceosomes
Extensive evidence has suggested that the nucleus is compartmentalized with respect to gene expression (for a review, see Spector, 2003). IGCs are enriched in pre-mRNA splicing factors, yet these nuclear regions are not sites of splicing or transcription. Rather, they are sites of splicing factor assembly/modification and/or storage (for a review, see Lamond and Spector, 2003) from which factors are recruited to nearby sites of active transcription. The C-terminal domain of the large subunit of RNA polymerase II and phosphorylation of the RS domain of SR splicing factors play a major role in supplying these factors to the site of active transcription (Misteli et al., 1998; Misteli and Spector, 1999). However, it has not been determined whether different splicing factors are targeted to a site of transcription individually, or as subcomplexes as needed for different stages of pre-mRNA processing. The latter is a possibility, because individual interchromatin granules are of a consistent size with ribosomes and are therefore large enough to contain such subcomplexes of proteins. When we made a comparison of protein components of the spliceosome (Zhou et al., 2002) versus IGC components, we found significant (63%), but not total overlap, between these two structures, although each complex was initially purified from an entirely distinct nuclear fraction.
Because there is considerable overlap of IGC components (modification/assembly and/or storage sites) with spliceosome components (functional sites), there is a possibility that interchromatin granules move from the IGCs to the site of active transcription, rather than each protein moving individually. It has been shown that fluorescently tagged splicing factors are highly mobile in living cells, but they move slowly enough to suggest that the proteins move in a complex, rather than as a monomer (Kruhlak et al., 2000). By time-lapse microscope analysis, it was shown that "spheres" seem to bud off of the surface of nuclear speckles when cells are actively transcribing (Eils et al., 2000). It remains to be determined whether these spheres correspond to an individual granule or clusters of IGC granules.
Apoptosis and Other Functions
In addition to proteins functioning in pre-mRNA splicing and transcription, we detected proteins that are involved in other nuclear functions. For example, acinus (KIAA0670) has been reported to be involved in a late step of an apoptotic pathway (Sahara et al., 1999). An in vitro system using permeabilized cells and apoptotic cell lysates revealed that acinus is activated by caspase 3 cleavage, and it induces apoptotic chromatin condensation in the absence of DNA fragmentation (Sahara et al., 1999). It was also shown that acinus is important for apoptotic chromatin condensation in vivo by using antisense RNA (Sahara et al., 1999). Recently, a complex called ASAP, containing RNPS1 (splicing factor), acinus and SAP18 (Sin3-associated protein; a component of a histone deacetylase complex), was isolated and the complex was shown to promote both pre-mRNA splicing and apoptosis, suggesting a possible link among apoptosis, splicing, and chromatin modification (Schwerk et al., 2003). Interestingly, acinus contains an RS domain (Boucher et al., 2001) that accounts for its localization to IGCs (Figure 2).
A second protein implicated in apoptosis, Btf (KIAA0164), was identified as a protein associated with the adenovirus oncoprotein E1B 19K as well as Bcl-2 family members. Btf has a transcriptional repression activity and its sustained overexpression induces apoptosis and suppresses transformation by E1A and E1B-19K or mutant p53 (Kasof et al., 1999). Although we have found that acinus colocalized within IGCs, Btf is localized at the periphery of IGCs (Figure 2).
As potential IGC proteins, we detected DNA repair proteins such as XPE UV-damaged DNA binding protein and XPA-binding protein 2 (Table 2). It is also interesting that we detected several types of "chaperone" proteins such as Hsp70, Dna J protein homolog, or RuvB like DNA helicase. In the developing kidney, Hsp70 is colocalized with Wilms tumor suppressor WT-1 in a speckled nuclear distribution pattern (Maheswaran et al., 1998). In the plant Brassica napus, it was shown that Hsp70 becomes associated with RNP structures in the interchromatin region and the nucleolus upon stress treatment to induce embryogenesis of microspores (Segui-Simarro et al., 2003). Although the localization of Hsp70 in IGCs remains to be confirmed, it would be interesting to analyze the changes in protein components in IGCs throughout the stages of development, oncogenesis, or environmental changes.
Recently, it has been suggested that transcription and translation are coupled. A small amount of translation, which might be important for quality control of gene products, has been reported to take place in the nucleus before export of mRNAs to the cytoplasm where the majority of translation occurs (Iborra et al., 2001). Thus far, we have detected two isoforms of eukaryotic initiation factor 4A, eIF4Ai and iii, in our proteomics analysis of IGCs. We and others also have found that fluorescently tagged eIF4Aiii is localized to IGCs (Holzmann et al., 2000). It has been shown that eIF4Ai, ii, and iii all confer RNA-dependent RNA helicase and ATP-dependent RNA helicase activities. However, they seem to function differently because eIF4Ai and ii facilitate translation, but eIF4Aiii inhibits translation in a reticulocyte lysate (Li et al., 1999). Recently, eIF4Aiii has been shown to be involved in nonsense-mediated decay (NMD) (Ferraiuolo et al., 2004). NMD is an RNA surveillance mechanism that serves to degrade mRNAs containing premature translation termination codons (for a review, see Maniatis and Reed, 2002; Wilkinson and Shyu, 2002; Singh and Lykke-Andersen, 2003). In our IGC fraction, we identified numerous members of the exon-exon junction complex that contains factors that are required for both mRNA export and NMD [Aly, RNPS1, RNA binding motif protein 8 (Y14), and mago-nashi homolog (MAGOH)]. This finding raises the possibility that proteins involved in these processes may be recruited from IGCs to transcription sites.
Motif Analysis
As expected, we detected many proteins with RNA binding motifs, RS motifs, and RNA helicase motifs, including ATP binding DEAD box helicases. However, thus far we have not detected a single sequence motif that is common among all IGC proteins. Therefore, aside from the RS domain, which serves to target certain proteins to IGCs, many other IGC-associated proteins may assemble into these structures by specific protein–protein and/or protein–RNA interactions rather than by a single targeting signal. Interestingly, 82% of the identified IGC proteins contain low complexity regions, such as a long stretch of a single type of amino acid, which could be involved in interactions with RNA or other proteins.
Because the RS motif seems to be unique among IGC proteins, we focused on a more in depth analysis of proteins containing an RS domain. We found that this group of proteins can be divided into several subgroups (Figure 3). In addition to the typical small RS domain-containing proteins that contain one or more RRMs, among which are members of the SR family of pre-mRNA splicing factors, there are larger RS domain-containing proteins containing additional domains and/or regions containing short repeats. It is plausible to imagine that these repeats are likely to perform a scaffolding function, as is found for certain HEAT repeat-containing proteins (Neuwald and Hirano, 2000). Also interesting are four proteins, U1 snRNP70, pre-mRNA cleavage factor Im, U1 small ribonucleoprotein 1, and acinus, that have degenerated RS domains in which the RS repeat itself contains, or is continuous with, RD/E dipeptides. RE repeats were previously found in the splicing factor YT521-B and were shown to be important for localization to the YT body, a subnuclear structure that is similar to but distinct from nuclear speckles (Nayler et al., 2000). The RD/E dipeptide motif is reminiscent of a phosphorylated RS domain, because the serine residue in RS is replaced with a negatively charged aspartic acid or glutamic acid. Interestingly, YT521-B was shown to localize to transcriptionally active sites and was suggested to play a role in grouping genes into higher order structures (Nayler et al., 2000). Thus, proteins with both RS and RD/E motifs may bridge sites of active transcription with IGCs.
In summary, we have characterized the proteome of IGCs purified from mouse liver nuclei. Although the protein identification supports a role of these nuclear domains in events relating to pre-mRNA processing, a significant number of new proteins have been identified, as well as interesting domains of known proteins. These will provide the impetus for future studies aimed at deciphering the organization and additional function(s) associated with this nuclear organelle.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Present address: Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto-shi, Kumamoto 860-0811, Japan. 
Corresponding author. E-mail addresss: spector{at}cshl.org.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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