Nuclear Matrix Fraction Research Articles

Replication Protein A and the Mre11·Rad50·Nbs1 Complex Co-localize and Interact at Sites of Stalled Replication Forks

In response to replicative stress, cells relocate and activate DNA repair and cell cycle arrest proteins such as replication protein A (RPA, a three subunit protein complex required for DNA replication and DNA repair) and the MRN complex (consisting of Mre11, Rad50, and Nbs1; involved in DNA double-strand break repair). There is increasing evidence that both of these complexes play a central role in DNA damage recognition, activation of cell cycle checkpoints, and DNA repair pathways. Here we demonstrate that RPA and the MRN complex co-localize to discrete foci and interact in response to DNA replication fork blockage induced by hydroxyurea (HU) or ultraviolet light (UV). Members of both RPA and the MRN complexes become phosphorylated during S-phase and in response to replication fork blockage. Analysis of RPA and Mre11 in fractionated lysates (cytoplasmic/nucleoplasmic, chromatin-bound, and nuclear matrix fractions) showed increased hyperphosphorylated-RPA and phosphorylated-Mre11 in the chromatin-bound fractions. HU and UV treatment also led to co-localization of hyperphosphorylated RPA and Mre11 to discrete detergent-resistant nuclear foci. An interaction between RPA and Mre11 was demonstrated by co-immunoprecipitation of both protein complexes with anti-Mre11, anti-Rad50, anti-NBS1, or anti-RPA antibodies. Phosphatase treatment with calf intestinal phosphatase or lambda-phosphatase not only de-phosphorylated RPA and Mre11 but also abrogated the ability of RPA and the MRN complex to co-immunoprecipitate. Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU- or UV-induced replication stress and suggest that protein phosphorylation may play a role in this interaction.

Nuclear diacylglycerol kinase-θ is activated in response to nerve growth factor stimulation of PC12 cells

Previous evidence from independent laboratories has shown that the nucleus contains diacylglycerol kinase (DGK) isoforms, i.e., the enzymes, which yield phosphatidic acid from diacylglycerol, thus terminating protein kinase C-mediated signaling events. A DGK isoform, which resides in the nucleus of PC12 cells, is DGK-θ. Here, we show that nerve growth factor (NGF) treatment of serum-starved PC12 cells results in the stimulation of both a cytoplasmic and a nuclear DGK activity. However, time course analysis shows that cytoplasmic DGK activity peaked earlier than its nuclear counterpart. While nuclear DGK activity was dramatically down-regulated by a monoclonal antibody known for selectively inhibiting DGK-θ, cytoplasmic DGK activity was not. Moreover, nuclear DGK activity was stimulated by phosphatidylserine, an anionic phospholipid that had no effect on cytoplasmic DGK activity. Upon NGF stimulation, the amount and the activity of DGK-θ, which was bound to the insoluble nuclear matrix fraction, substantially increased. Epidermal growth factor up-regulated a nuclear DGK activity insensitive to anti-DGK-θ monoclonal antibody. Overall, our findings identify nuclear DGK-θ as a down-stream target of NGF signaling in PC12 cells.

Expression and functional significance of mouse paraspeckle protein 1 on spermatogenesis.

Paraspeckle protein 1 (PSP1) in humans is a recently identified component protein of a novel nuclear body, paraspeckle. The protein has a DBHS (Drosophila behavior, human splicing) motif that is found in PSF and p54(nrb)/NonO proteins. These DBHS-containing proteins have been reported to be involved in various nuclear events such as DNA replication, transcription, and mRNA processing. Here we show that mouse paraspeckle protein 1 (mPSP1; encoded by the Pspc1 gene) has two isoforms with different C-termini lengths. Abundant expression of the longer isoform (mPSP1-alpha) and the shorter one (mPSP1-beta) were observed in testis and kidney, respectively. Transiently expressed mPSP1-alpha was localized in nuclei, but mPSP1-beta was localized in both nuclei and cytoplasm. These observations suggest that alternative splicing regulates tissue distribution and subcellular localization. Like other DBHS-containing proteins, mPSP1 has RNA-binding activity. In mouse testis, mPSP1-alpha was found in the nuclear matrix fraction. Furthermore, by coimmunoprecipitation, we confirmed that mPSP1 interacts with other DBHS-containing proteins, PSF and p54(nrb)/NonO. Therefore, we conclude that mPSP1 may regulate multiple phases of important nuclear events during spermatogenesis.

Transcriptional coregulator SNW/SKIP: the concealed tie of dissimilar pathways.

Eukaryotic gene expression requires that all the steps of messenger RNA production are regulated in concert to integrate the diverse inputs cells receive. We discuss the functioning of SNW/SKIP, an essential spliceosomal component and transcriptional coregulator, which may provide regulatory coupling of transcription initiation and splicing. SNW/SKIP potentiates the activity of important transcription factors, such as vitamin D receptor, CBF1 (RBP-Jkappa), Smad2/3, and MyoD. It synergizes with Ski in overcoming pRb-mediated cell cycle arrest, and it is targeted by the viral transactivators EBNA2 and E7. SNW/SKIP may aid in conformational transition of the gene expression machine through its avidity to nuclear matrix fractions or by recruiting foldases such as the prolyl isomerase PPIL1. The extensive list of SNW/SKIP partners, its unique primary structure, conserved from yeast to humans, and its essential character suggest a distinct function of general importance.

Proteasome-dependent dispersal of PML nuclear bodies in response to alkylating DNA damage.

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are present in variable number in most human cell types and have been linked to various cellular functions, including roles as depots for DNA repair proteins. Here, we show that treatment of human cells with DNA methylating agents leads to redistribution of PML from NBs to a diffuse nuclear localization. Biochemically, this correlates with a specific reduction of PML levels in the nuclear matrix fraction without affecting total PML levels. Similar results were obtained for the other major PML NB component, the Sp100 protein, indicating that DNA methylating agents lead to a general disassembly of PML NBs. Similar to the dispersal of PML NBs in response to some viral infections, PML redistribution after DNA damage was inhibited by the proteasome inhibitor MG132. We propose that the regulated dispersal of PML NBs may facilitate the enhanced release of DNA repair proteins from NB depots in order to respond adequately to extensive DNA damage.

Genomic imprinting controls matrix attachment regions in the Igf2 gene.

Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.

Nuclear matrix bound fibroblast growth factor receptor is associated with splicing factor rich and transcriptionally active nuclear speckles.

We have used confocal microscopy combined with computer image analysis to evaluate the functional significance of a constitutively expressed form of the receptor tyrosine kinase FGFR1 (fibroblast growth factor receptor 1) in the nucleus of rapidly proliferating serum stimulated TE 671 cells, a medullobastoma human cell line. Our results demonstrate a limited number of large sites and numerous smaller sites of FGFR1 in the nuclear interior. The larger sites showed virtually complete colocalization (>90%) with splicing factor rich nuclear speckles while the smaller sites showed very limited overlap (<20%). Similar results were found for several other proliferating cell lines grown in culture. An in situ transcription assay was used to determine colocalization with transcription sites by incorporating 5-bromouridine triphosphate (BrUTP) followed by dual staining for BrUTP and FGFR1. These results combined with those from using an antibody against the large subunit of RNA polymerase II suggest a significant degree of colocalization (26-38%) over both the large and small sites. No colocalization was detected with sites of DNA replication. The spatial arrangements of FGFR1 sites and colocalization with nuclear speckles were maintained following extraction for nuclear matrix. Moreover, immunoblots indicated a significant enrichment of FGFR1 in the nuclear matrix fraction. Our findings suggest an involvement of a nuclear matrix bound FGFR1 in transcriptional and RNA processing events in the cell nucleus. We further propose that nuclear speckles, aside from a role in transcriptional/RNA processing events, may serve as fundamental regulatory factories for the integration of diverse signaling and regulatory factors that impact transcription and cellular regulation.

Epstein-Barr virus nuclear antigen-1 colocalizes with lamin B1 in the nucleoplasm and along the nuclear rim.

Epstein-Barr virus nuclear antigen 1 (EBNA-1) is essential for the maintenance of latent EBV plasmids, and is also a transcriptional regulator. Nuclear lamins, components of the nuclear lamina, have also been found in the nucleoplasm. We report here that EBNA-1 coincided with lamin B1 in the nucleoplasm and around the nuclear rim during S-phase by confocal microscopy of cells transfected with EBNA-1 in the absence of EBV plasmids. Lamin B1, which is rarely detected in nuclear soluble fractions, was detected in chromatin and nuclear matrix fractions of the EBNA-1-expressing cells. These observations suggest that EBNA-1 colocalizes with lamin B1 in the subnuclear sites.

Diacylglycerol kinase-θ is localized in the speckle domains of the nucleus

It is well established that the nucleus is endowed with enzymes that are involved in lipid-dependent signal transduction pathways. Diacylglycerol (DAG) is a fundamental lipid second messenger that is produced in the nucleus. Previous reports have shown that the nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting DAG into phosphatidic acid (PA), terminate DAG-dependent events. Here, we show, by immunofluorescence staining and confocal analysis, that DGK-θ localizes mainly to the nucleus of various cell lines, such as MDA-MB-453, MCF-7, PC12, and HeLa. Nuclear DGK-θ co-localizes with phosphatidylinositol 4,5-bisphosphate (PIP 2) in domains that correspond to nuclear speckles, as revealed by the use of an antibody to the splicing factor SC-35, a well-established marker for these structures. The spatial distribution of nuclear DGK-θ was dynamic in that it was affected by inhibition of mRNA transcription with α-amanitin. Immuno-electron microscopy analysis demonstrated that DGK-θ, PIP 2, and phosphoinositide-specific phospholipase Cβ1 (PLCβ1) associated with electron-dense particles within the nucleus that correspond to interchromatin granule clusters. Cell fractionation experiments performed in MDA-MB-453, HeLa, and PC12 cells showed a preferential association of DGK-θ with the nucleus. Western blots demonstrated that DGK-θ was enriched in the nuclear matrix fraction prepared from MDA-MB-453 cells. Immunoprecipitation experiments with an antibody to PLCβ1 revealed in MDA-MB-453 cells an association between this enzyme and both DGK-θ and phosphatidylinositol phosphate kinase Iα (PIPKIα). Our findings strengthen the contention that speckles represent a crucial site for the nuclear-based inositol lipid cycle. We may speculate that nuclear speckle-located DGK-θ, on cell stimulation with an agonist, converts to PA the DAG derived from PLCβ1-dependent PIP 2 hydrolysis.

A novel alpha-helical protein, specific to and highly conserved in plants, is associated with the nuclear matrix fraction.

A cDNA for a novel plant protein was isolated from tomato. Nuclear Matrix Protein 1 (NMP1) is a ubiquitously expressed 36 kDa protein, which has no homologues in animals and fungi, but is highly conserved among flowering and non-flowering plants, including gymnosperms, moss, and the liverwort Marchantia polymorpha. NMP1 is predominantly alpha-helical with multiple stretches of short amphipathic regions. Cell fractionation, immunofluorescence, and GFP localization experiments showed that NMP1 is located both in the cytoplasm and nucleus and that the nuclear fraction is associated with the nuclear matrix. NMP1 is a candidate for a plant-specific structural protein with a function both in the nucleus and cytoplasm.

Cell cycle dependent regulation of protein kinase CK2 signaling to the nuclear matrix.

Protein kinase CK2 is a ubiquitous protein serine/threonine kinase that is involved in cell growth and proliferation as well as suppression of apoptosis. Several studies have suggested that the kinase plays a role in cell cycle progression; however, changes in enzyme activity during phases of cell cycle have not been detected. Nuclear matrix is a key locus for CK2 signaling in the nucleus. We therefore examined CK2 signaling to the nuclear matrix in distinct phases of cell cycle by employing synchronized ALVA-41 prostate cancer cells. Removal of serum from the culture medium resulted in G0/G1 arrest, and a reduction in the nuclear matrix-associated CK2 activity which was rapidly reversed on addition of serum. Arresting the cells in G(0)/G(1) phase with hydroxyurea and subsequent release to S phase by serum gave similar results. Cells arrested in the G(2)/M phase by treatment with nocodazole demonstrated an extensive reduction in the nuclear matrix-associated CK2 which was reversed rapidly on addition of serum. Changes in the immunoreactive CK2 protein were concordant with the activity data reflecting a dynamic trafficking of the kinase in distinct phases of cell cycle. Under the same conditions, CK2 activity in total cellular lysate remained essentially unaltered. These results provide the first direct evidence of discrete modulations of CK2 in the nuclear matrix during the cell cycle progression. Inducible overexpression of CK2 in CHO cells yielded only a modest increase in CK2 activity even though a significant increase in expression was apparent at the level of CK2 alpha-specific message. Stably transfected ALVA-41 cells, however, did not show a significant change in CK2 levels despite increased expression at the message level. Not surprisingly, both types of the stably transfected cells failed to show any alteration in cell cycle progression. Distribution of the CK2 activity in the cytosolic versus nuclear matrix fractions in normal cells appears to be different from that in the cancer cells such that the ratio of nuclear matrix to cytosolic activity is much higher in the latter. Considering that nuclear matrix is central to several nuclear functions, this pattern of intracellular distribution of CK2 may have implications for its role in the oncogenic process. Published 2003 Wiley-Liss, Inc.

The controversial nuclear matrix: a balanced point of view.

The nuclear matrix is defined as the residual framework after the removal of the nuclear envelope, chromatin, and soluble components by sequential extractions. According to several investigators the nuclear matrix provides the structural basis for intranuclear order. However, the existence itself and the nature of this structure is still uncertain. Although the techniques used for the visualization of the nuclear matrix have improved over the years, it is still unclear to what extent the isolated nuclear matrix corresponds to an in vivo existing structure. Therefore, considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the situation in living cells. Here, we summarize the experimental evidence in favor of, or against, the presence of a diffuse nucleoskeleton as a facilitating organizational nonchromatin structure of the nucleus.

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a′ domain

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a ′ domain as directly involved in the DNA-binding activity of ERp57.

The Role of Topoisomerase II in the Excision of DNA Loop Domains during Apoptosis

Disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal DNA fragments represents two major periodicities of DNA fragmentation during apoptosis. These are thought to originate from the excision of DNA loop domains and from the cleavage of nuclear DNA at the internucleosomal positions, respectively. In this report, we demonstrate that different apoptotic insults induced apoptosis in NB-2a neuroblastoma cells that was invariably accompanied by the formation of HMW DNA fragments of about 50-100 kb but proceeded either with or without oligonucleosomal DNA cleavage, depending on the type of apoptotic inducer. We demonstrate that differences in the pattern of DNA fragmentation were reproducible in a cell-free apoptotic system and develop conditions that allow in vitro separation of the HMW and oligonucleosomal DNA fragmentation activities. In contrast to apoptosis associated with oligonucleosomal DNA fragmentation, the HMW DNA cleavage in apoptotic cells was accompanied by down-regulation of caspase-activated DNase (CAD) and was not affected by z-VAD-fmk, suggesting that the caspase/CAD pathway is not involved in the excision of DNA loop domains. We further demonstrate that nonapoptotic NB-2a cells contain a constitutively present nuclease activity located in the nuclear matrix fraction that possessed the properties of topoisomerase (topo) II and was capable of reproducing the pattern of HMW DNA cleavage that occurred in apoptotic cells. We demonstrate that the early stages of apoptosis induced by different stimuli were accompanied by activation of topo II-mediated HMW DNA cleavage that was reversible after removal of apoptotic inducers, and we present evidence of the involvement of topo II in the formation of HMW DNA fragments at the advanced stages of apoptosis. The results suggest that topo II is involved in caspase-independent excision of DNA loop domains during apoptosis, and this represents an alternative pathway of apoptotic DNA disintegration from CAD-driven caspase-dependent oligonucleosomal DNA cleavage.

Small heat shock protein p26 associates with nuclear lamins and HSP70 in nuclei and nuclear matrix fractions from stressed cells

The small heat shock/alpha-crystallin protein p26 undergoes nuclear translocation in response to stress in encysted embryos of the brine shrimp Artemia franciscana. About 50% of total p26 translocates to nuclei in embryos treated with heat shock or anoxia, and in embryo homogenates incubated at low pH. Nuclear fractionation shows that the majority of nuclear p26 and a nuclear lamin are associated with the nuclear matrix fraction. To further explore the roles of p26 and other HSPs in stabilizing nuclear matrix proteins (NMPs), nuclear matrices from control, and heat-shocked embryos were disassembled in urea and evaluated by one and two-dimensional (2-D) gel electrophoresis and Western immunoblotting after reassembling. Nuclear lamins were present only in reassembled fractions and, in the case of heat shock, p26 and HSP70 were also present. HSP90 was not detected in any nuclear fraction. Confocal microscopy on isolated nuclei and nuclear matrix preparations from control and heat-shocked embryos showed that the majority of p26 and a nuclear lamin share similar nuclear distributions. The combination of microscopy and fractionation results suggests that p26 and HSP70 play a role in the protection of nuclear lamins within the nuclear matrix.

Hsp27 protects the cytoskeleton and nucleus from the effects of 42°C at pH 6.7 in CHO cells adapted to growth at pH 6.7

CHO cells are normally sensitized to hyperthermia by acidic pH. However, CHO cells adapted to growth in pH 6.7 medium become less sensitive to heat killing at the reduced pH. The adapted cells maintain their ability to develop thermotolerance at pH 6.7 and their steady state intracellular pH is elevated. Furthermore, the small molecular weight stress chaperone, hsp27, is elevated in unheated cells maintained at pH 6.7. This report documents that the cytoskeletal and nuclear components of the low pH adapted CHO cells are resistant to 42°C-induced collapse and protein accretion, respectively. Hyperthermia induced a perinuclear collapse of the microtubular cytoskeleton and an increase in the amount of insoluble protein associated with the nuclei and nuclear matrix fractions in the control cells heated at pH 7.3 or heated after acute acidification to pH 6.7. Protection from these effects was observed in the low pH adapted cells heated at pH 6.7. Hsp70 does not appear to play a dominant role in the response of the adapted cells to 42°C. The induction of hsp70 during heating is abrogated by pH 6.7 in cells cultured at either pH 7.3 or pH 6.7. The resistance of the microtubular cytoskeleton to perinuclear collapse and the absence of protein aggregation in the nucleus during 42°C may be due to the elevated levels of hsp27 both before heating and during the heat treatment. In summary, the phenotype of CHO cells adapted to growth at low pH includes resistance of the cytoskeleton to 42°C-induced perinuclear collapse and resistance to 42°C-induced aggregation of nuclear proteins, in addition to the reduction in heat cytotoxicity, upregulation of intracellular pH and upregulation of hsp27.

Small heat shock protein p26 associates with nuclear lamins and HSP70 in nuclei and nuclear matrix fractions from stressed cells

Small heat shock protein p26 associates with nuclear lamins and HSP70 in nuclei and nuclear matrix fractions from stressed cells

The Matrix Attachment Region-binding Protein SATB1 Interacts with Multiple Elements within the gp91phox Promoter and Is Down-regulated during Myeloid Differentiation

The gp91(phox) gene encodes a component of the respiratory burst NADPH oxidase complex and is highly expressed in mature myeloid cells. The transcriptional repressor CCAAT displacement protein binds to at least five sites within the proximal gp91(phox) promoter and represses expression prior to terminal phagocyte differentiation. The DNA binding activity of CCAAT displacement protein decreases during terminal phagocyte differentiation, thus permitting the binding of transcriptional activators and induction of gp91(phox) expression. We report here that the matrix attachment region-binding protein SATB1 interacts with at least seven sites within the -1542 to +12-base pair gp91(phox) promoter. Four additional binding sites for CCAAT displacement protein were also identified. Furthermore, the most proximal SATB1-binding site within the gp91(phox) promoter binds specifically to the nuclear matrix fraction in vitro. SATB1 expression is down-regulated during terminal myeloid cell differentiation, coincident with induction of gp91(phox) expression. Transient transfection assays demonstrate that a SATB1-binding site derived from the gp91(phox) promoter represses promoter activity in cells expressing SATB1. These findings underscore the importance of transcriptional repression in the regulation of gp91(phox) expression and reveal a candidate myeloid cell target gene for SATB1, a factor previously found to be essential for T cell development.

Fanconi Anemia Proteins Localize to Chromatin and the Nuclear Matrix in a DNA Damage- and Cell Cycle-regulated Manner

Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix.

Localization and Stability of Introns Spliced from thePem Homeobox Gene

RNA splicing generates two products in equal molar amounts, mature mRNAs and spliced introns. Although the mechanism of RNA splicing and the fate of the spliced mRNA products have been well studied, very little is known about the fate and stability of most spliced introns. Research in this area has been hindered by the widely held view that most vertebrate introns are too unstable to be detectable. Here, we report that we are able to detect all three spliced introns from the coding region of the Pem homeobox gene. By using a tetracycline (tet)-regulated promoter, we found that the half-lives of these Pem introns ranged from 9 to 29 min, comparable with those of short lived mRNAs such as those encoding c-fos and c-myc. The half-lives of the Pem introns correlated with both their length and 5' to 3' orientation in the Pem gene. Subcellular fractionation analysis revealed that spliced Pem introns and pre-mRNA accumulated in the nuclear matrix, high salt-soluble, and DNase-sensitive fractions within the nucleus. Surprisingly, we found that all three of the spliced Pem introns were also in the cytoplasmic fraction, whereas Pem pre-mRNAs, U6 small nuclear RNA, and a spliced intron from another gene were virtually excluded from this fraction. This indicates either that spliced Pem introns are uniquely exported to the cytoplasm for degradation or they reside in a unique soluble nuclear fraction. Our study has implications for understanding the regulation of RNA metabolism, as the stability of introns and the location of their degradation may dictate the following: (i) the stability of nearby mRNAs that compete with spliced introns for rate-limiting nucleases, (ii) the rate at which free nucleotides are available for further rounds of transcription, and (iii) the rate at which splicing factors are recycled.