Perichromatin Fibrils Research Articles

Electron microscopy of nuclear nanoribonucleoproteins (nanoRNPs)

In the cell nucleus several ribonucleoproteins related to gene expression are present The nucleolus coiled bodies and other nuclear particles are among these structures Particles of nanometer size include perichromatin fibrils perichromatin and Balbiani ring granules interchromatin granules and Lacandonia granules All of them are involved in pre messenger RNA intranuclear metabolism Previous and recent research suggests that these particles are involved in transcription and processing of pre messenger RNA within the cell nucleus

Isolation and Identification of Gene-Specific MicroRNAs.

Computer programming has identified hundreds of genomic hairpin sequences, many with functions yet to be determined. Because transfection of hairpin-like microRNA precursors (pre-miRNAs) into mammalian cells is not always sufficient to trigger RNA-induced gene silencing complex (RISC) assembly, a key step for inducingRNA interference (RNAi)-related gene silencing, we havedeveloped an intronic miRNA expressionsystem to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene, and hencesuccessfully increase the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis mechanismhas been found to depend on a coupled interaction of nascent messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNAso obtained is transcribed by type-II RNApolymerases, coexpressed within a primary gene transcript, and thenexcised out of the gene transcript by intracellular RNA splicing and processing machineries. After that, ribonuclease III (RNaseIII)endonucleases further process the spliced introns into mature miRNAs. Using this intronicmiRNA expression system, we have shown for the first time that the intron-derived miRNAs are able to elicit strong RNAi effects in not only human and mouse cells in vitrobut also in zebrafishes, chicken embryos, and adult micein vivo. We have also developed a miRNA isolation protocol, based on the complementarity between the designed miRNA and its targeted gene sequence, to purify and identify the mature miRNAs generated. As a result, several intronic miRNA identities and structures have been confirmed. According to this proof-of-principle methodology, we now have full knowledge to design variousintronicpre-miRNA inserts that are more efficient and effective for inducing specific gene silencing effects in vitro and in vivo.

Unexpected distribution of KRIT1 inside the nucleus: new insight in a complex molecular pathway

KRIT1 is an 84kDa protein that lacks any relevant catalytic domains, associated with the cerebral cavernous malformation disease. We have investigated by means of ultrastructural immunocytochemistry the nuclear distribution of KRIT1 in different cell lines, revealing its unexpected localization on actively transcribing nuclear domains such as the perichromatin fibrils and the nucleolar dense fibrillar component. These preliminary data indicate a still undescribed and unknown role for KRIT1 inside the nucleus.

Friend of Prmt1, FOP is a Novel Component of the Nuclear SMN Complex Isolated Using Biotin Affinity Purification

SMN (survival motor neuron protein) complexes are essential for the biogenesis of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). During the biogenesis, the SMN complexes bound to UsnRNPs are transported from the cytoplasm to the nucleus, and moved to Cajal body (bodies)/Gems (Cajal/Gems) where the SMN complexes- UsnRNPs are subjected to additional chemical modifications and dissociated to the SMN complexes and the mature UsnRNPs. Although the mature UsnRNPs are assembled into spliceosome with newly transcribed pre-mRNA in the perichromatin fibrils at the chromatin, the role of the dissociated nuclear SMN complexes remains undetermined. In this study, we identified Friend of Prmt1 (FOP; chromatin target of Prmt1, CHTOP; C1orf77) as a novel component of the nuclear SMN complexes by the biotin affinity purification, coupled with the mass spectrometry-based protein identification. FOP was associated with SMN, Gemines 2, 3, 4, 6, and 8, unrip, and fragile X mental retardation 1 protein (FMR1), as well as with U5and U6 snRNAs in the nucleus, but not with Sm proteins, Gemin5, coilin, and U1 and U2snRNAs. Using the quantitative proteomic method with SILAC coupled with RNA interference, we also showed that FOP is required for the association of the SMN complexes with hnRNPs, histone proteins, and various RNA-binding proteins. It is reported that FOP localizes mainly in the nuclear speckles, binds chromatin, and plays a role in mRNA transcriptional regulation. Our present data suggest that the nuclear SMN complex containing FOP participates in the process of mRNA post-transcriptional regulation.

Perispeckles are major assembly sites for the exon junction core complex

The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear speckles. Fluorescence resonance energy transfer analyses and EJC assembly mutants show that perispeckles do not store free subunits, but instead are enriched for assembled cores. At the ultrastructural level, perispeckles are distinct from interchromatin granule clusters that may function as storage sites for splicing factors and intermingle with perichromatin fibrils, where nascent RNAs and active RNA Pol II are present. These results support a model in which perispeckles are major assembly sites for the tetrameric EJC core. This subnuclear territory thus represents an intermediate region important for mRNA maturation, between transcription sites and splicing factor reservoirs and assembly sites.

A change of developmental program induces the remodeling of the interchromatin domain during microspore embryogenesis in Brassica napus L.

After a stress treatment, in vitro-cultured pollen changes its normal gametophytic developmental pathway towards embryogenesis producing multicellular embryos from which, finally, haploid and double haploid plants develop. The architecture of the well-organized nuclear functional domains changes in response to DNA replication, RNA transcription, processing and transport dynamics. A number of subnuclear structures present in the interchromatin region (IR, the nuclear domain between chromosome territories) have been shown as involved, either directly or indirectly, in transcriptional regulation. These structures include the interchromatin granule clusters (IGCs), perichromatin fibrils (PFs), Cajal bodies (CBs) and perichromatin granules (PGs). In this work, we present a cytochemical, immunocytochemical, quantitative and morphometric analysis at the light, confocal and electron microscopy levels to characterize the changes in the functional architecture of the nuclear interchromatin domain during two developmental programs followed by the microspore: differentiation to mature pollen grains (transcriptionally inactive), and microspore embryogenesis involving proliferation in the first stages (highly engaged in transcription). Our results revealed characteristic changes in size, shape and distribution of the different interchromatin structures as a consequence of the reprogramming of the microspore, allowing us to relate the remodeling of the interchromatin domain to the variations in transcriptional activities during proliferation and differentiation events, and suggesting that RNA-associated structures could be a regulatory mechanism in the process. In addition, we document the presence of two structurally different types of CBs, and of IGC and CB-associated regions, similar to those present in animal cells, and not yet described in plants.

Perichromatin Fibrils Accumulation in Hepatocyte Nuclei Reveals Alterations of Pre-mRNA Processing During Aging

We previously described an unusual accumulation of perichromatin fibrils (PF)-the in situ form of pre-mRNA transcription and early splicing-in hepatocyte nuclei of old rats. Here we have investigated, by immunoelectron microscopy, the nature of such PF, analyzing the presence of transcription, splicing and cleavage factors, polyadenylated RNA, and the incorporation of bromouridine in adult and old rats. Our observations revealed alterations in amount and/or distribution of pre-mRNA transcription, splicing and cleavage factors, as well as of polyadenylated RNA, together with lower bromouridine incorporation in newly transcribed RNA in the hepatocyte nucleoplasm of old rats. Therefore, our data indicate both a decrease in pre-mRNA transcription and a slow down of PF processing and transport during aging.

Immunocytochemical study of estrogen receptor activation factor (E-RAF) and the proteins that interact with nuclear estrogen receptor II (nER II) in epithelial endometrial cells, in the presence and in the absence of estradiol

The localization and abundance of the estrogen receptor activation factor (E-RAF) and a small nuclear ribonucleoprotein (snRNP) complex containing three proteins, p32, p55 and p60, which interact with the nuclear estrogen receptor II (nER II), have been studied in rat endometrial epithelial cells by means of immunofluorescence and high resolution quantitative immunocytochemistry. In the cytoplasm E-RAF is associated with the rough endoplasmic reticulum. In the nucleus it is mainly localized at the interchromatin space, and surrounding the clumps of compact or semi-condensed chromatin. Quantitative analyses show that the abundance of E-RAF in the nucleus increases after ovariectomy and decreases 3 minutes after estradiol administration. These results are in agreement with the currently available biochemical data. Double immunolocalizations demonstrate that p32, p55, p60 co-localize with other splicing-related protein. High resolution immunolocalization shows that p32, p55, p60 are associated with perichromatin fibrils (co-transcriptional splicing) and with clusters of interchromatin granules (storage of splicing-related molecules). The nuclear abundance of the snRNP complex decreases with ovariectomy, increases within 3 minutes after estradiol administration and remains higher than that in ovariectomized animals for 27 minutes. These results strongly support the previous data on the role of nER-II in the regulation of mRNA transcription and its export from the nucleus to the cytoplasm.

Fine distribution of CLOCK protein in hepatocytes of hibernating dormice.

CLOCK protein is a member of the bHLH-PAS family of transcription factors, it is expressed in several tissues including the liver and is essential for normal circadian rhythms. In this study we investigate the distribution of CLOCK protein in hepatocytes of euthermic and hibernating edible dormice Glis glis as well as in hepatocytes taken from the hibernating animals submitted in vitro to experimental conditions mimicking the arousal process. Our results demonstrate that CLOCK protein is expressed in all animals and is mostly located in the nucleus, in particular, on perichromatin fibrils and nucleoli. During deep hibernation CLOCK protein becomes more abundant but an intracellular redistribution occurs: the protein significantly decreases in all cellular compartments, but it accumulates in the amorphous bodies. These nuclear bodies, typical of the hibernating state, probably represent storage sites for CLOCK protein to be quickly used upon arousal. Accordingly, in hepatocytes submitted to in vitro conditions mimicking arousal CLOCK protein levels rapidly reach the euthermic values, while amorphous bodies disappear.

Visualization of transcription sites at the electron microscope.

In order to localize at EM level the sites of transcription of both pre-mRNA and pre-rRNA, we have detected the DNA/RNA hybrid molecules and m3Gcapped structures by means of specific antibodies after short bromo-uridine (BrU) incorporation. In addition, the sections have been stained by a selective RNA stain, terbium citrate. Our data indicate that perichromatin fibrils incorporate BrU and are labeled by the anti-hybrid probe; this supports the idea that they are the pre-mRNA transcription sites. On the contrary, interchromatin granules do not incorporate BrU after short pulses and are not labeled by the anti-hybrid probe. Concerning the nucleolus, anti-hybrid and anti-BrdU antibodies colocalize only on the dense fibrillar component, suggesting that this is the site of rRNA transcription. Interestingly, the dense fibrillar component and the granular component, after specific RNA staining, show remarkable structural similarities, both containing fibrogranular RNA structures.

TDP-43 localizes in mRNA transcription and processing sites in mammalian neurons

TDP-43 is a RNA/DNA-binding protein structurally related to nuclear hnRNP proteins. Previous biochemical studies have shown that this nuclear protein plays a role in the regulation of gene transcription, alternative splicing and mRNA stability. Despite the ubiquitous distribution of TDP-43, the growing list of TDP-43 proteinopathies is primarily associated with neurodegenerative disorders. Particularly, TDP-43 redistributes to the cytoplasm and forms pathological inclusions in amyotrophic lateral sclerosis and several forms of sporadic and familiar frontotemporal lobar degeneration. Here, we have studied the nuclear compartmentalization of TDP-43 in normal rat neurons by using light and electron microscopy immunocytochemistry with molecular markers for nuclear compartments, a transcription assay with 5'-fluorouridine, and in situ hybridization for telomeric DNA. TDP-43 is concentrated in euchromatin domains, specifically in perichromatin fibrils, nuclear sites of transcription and cotranscriptional splicing. In these structures, TDP-43 colocalizes with 5'-fluorouridine incorporation sites into nascent pre-mRNA. TDP-43 is absent in transcriptionally silent centromeric and telomeric heterochromatin, as well as in the Cajal body, a transcription free nuclear compartment. Furthermore, a weak TDP-43 immunolabeling is found in nuclear speckles of splicing factors. The specific localization of TDP-43 in active sites of transcription and cotranscriptional splicing is consistent with biochemical data indicating a role of TDP-43 in the regulation of transcription and alternative splicing.

S1‐1 nuclear domains: characterization and dynamics as a function of transcriptional activity

The RNA-binding protein S1-1, also called RBM10 (RNA-binding motif 10), is a paralogue of putative tumour suppressor RBM5 and has been correlated with cancer proliferation and apoptosis. In the present study, we have investigated the cell biology of S1-1. In the extranucleolar nucleoplasm, S1-1 occurred in hundreds of punctate and irregular domains. Some 10-40 of these domains were larger than 0.5 mum and prominent for S1-1 immunostaining. These domains (S1-1 nuclear bodies) were commonly present in tissue cells and in cultured cells. When cellular transcription was globally reduced by heat shock, serum starvation, culture at high cell densities or inhibition with RNA polymerase II inhibitors, small S1-1 domains (S1-1 granules), with weak immunostaining signals, reduced in number, whereas S1-1 nuclear bodies became prominent and increased in size. These altered S1-1 domains were returned to initial states when the cells were placed under normal conditions. Similar to paraspeckles, S1-1 nuclear bodies occurred closely adjacent to nuclear speckles or IGCs (interchromatin granule clusters), as determined by immunoelectron microscopy. However, the S1-1 nuclear bodies did not correspond to paraspeckles or IGAZs (interchromatin-granule-associated zones), but coincided with TIDRs (transcription-inactivation-dependent RNA domains), which we had characterized previously at the RNA level. The enlarged S1-1 nuclear bodies/TIDRs accumulated the S1-1 protein and microinjected primary and spliced mRNAs, presumably for later elevation of gene expression. In addition, electron microscopy revealed that S1-1 was also present on perichromatin fibrils, suggesting the structure of S1-1 granules seen at higher resolution. S1-1 constitutes hundreds of nuclear domains, which dynamically change their structures in a reversible manner. Upon globally reducing RNA polymerase II transcription, S1-1 nuclear bodies enlarge and decrease in number. They are novel domains different from paraspeckles or IGAZs, despite their similar occurrence adjacent to nuclear speckles. We discuss S1-1 granules in terms of their association with gene expression. In addition, this is the first report of a TIDR-localized protein.

Dynamic relocation of nuclear proteins during the execution phase of apoptosis

In the apoptotic program of controlled cell dismantling, the most characteristic nuclear changes involve chromatin, which condenses and often collapses against the nuclear envelope in the form of crescents. A severe reorganization also occurs in ribonucleoprotein (RNP)-containing structures which are involved in the synthesis and processing of transcripts: already during early apoptosis, the nucleoplasmic RNPs (namely, perichromatin fibrils, perichromatin granules, and interchromatin granules) coalesce in the interchromatin space where they associate with segregated nucleolar components, to ectopically form fibro-granular heterogeneous clusters. This was found to occur in cell systems in vivo and in cultured cell lines, after different apoptogenic stimuli. These RNP aggregates we have called heterogeneous ectopic RNP-derived structures (HERDS) move from the nucleus to the cytoplasm, and may be found in apoptotic bodies, in late apoptosis. Immunolabeling experiments demonstrated that several other proteins which are normally located inside the nucleus also move into the cytoplasm, during apoptosis, independently from HERDS. Apoptotic cells have been suggested to be a powerful source of nuclear auto-antigens, which are produced by the partial proteolytic or nucleolytic cleavage of a wide variety of nuclear substrates. In the presence of defective phagocytosis (or when massive apoptosis overwhelms the clearance capability of the tissue scavenger cells), the disposal of apoptotic cells becomes insufficient and unphagocytosed late apoptotic cells may accumulate in the tissue where they may be engulfed by antigen-presenting cells (such as dendritic cells); an autoimmune response may thus be elicited, by which apoptosis-derived auto-antigens are recognized and presented to the immune system.

Subnuclear localization and dynamics of the Pre-mRNA 3' end processing factor mammalian cleavage factor I 68-kDa subunit.

Mammalian cleavage factor I (CF Im) is an essential factor that is required for the first step in pre-mRNA 3' end processing. Here, we characterize CF Im68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF Im68 accumulates in structures that partially overlap with nuclear speckles. Analysis of synchronized cells shows that CF Im68 distribution in speckles and paraspeckles varies during the cell cycle. At an ultrastructural level, CF Im68 is associated with perichromatin fibrils, the sites of active transcription, and concentrates in interchromatin granules-associated zones. We show that CFIm68 colocalizes with bromouridine, RNA polymerase II, and the splicing factor SC35. On inhibition of transcription, endogenous CF Im68 no longer associates with perichromatin fibrils, but it can still be detected in interchromatin granules-associated zones. These observations support the idea that not only splicing but also 3' end processing occurs cotranscriptionally. Finally, fluorescence recovery after photobleaching analysis reveals that the CF Im68 fraction associated with paraspeckles moves at a rate similar to the more dispersed molecules in the nucleoplasm, demonstrating the dynamic nature of this compartment. These findings suggest that paraspeckles are a functional compartment involved in RNA metabolism in the cell nucleus.

Nuclear structure in early mouse embryos: A comparative ultrastructural and immunocytochemical study with special emphasis on the “2-cell block in vitro”

The embryos from many outbred and inbred strains of mice are arrested at the late 2-cell stage when cultured in vitro in simple culture media. This phenomenon is referred to as the “2-cell block in vitro”. The ultrastructural morphology of the nuclei of the blocked embryos is not yet well described. In the present paper we documented the results of a comparative study on the nuclei of mouse embryos, both normally developing and arrested at the 2-cell stage. The blocked embryos maintain the morphological integrity of their nuclei. Main nuclear domains (nucleolus precursor bodies, interchromatin granule clusters, perichromatin granules, and perichromatin fibrils), typical for the control embryos, are observed in the blocked ones. A number and morphological characteristics of these nuclear substructures are not changed significantly in the blocked embryos. At the same time, RNA polymerase II and pre-mRNA splicing factors are redistributed in the nucleus of the blocked embryos. Although something goes to show that nuclear organization of the blocked embryos differ from that of the control, we could not reveal in the blocked embryos distinct signs of degeneration which might characterize aged or dying cells. Our data confirm a peculiar functional state of the 2-cell blocked embryos.

The effect of the enkephalin DADLE on transcription does not depend on opioid receptors

[D-Ala(2),D-Leu(5)] enkephalin (DADLE) is a synthetic peptide capable of inducing a hibernation-like state in mammals in vivo and in cultured cells in vitro. The effects of DADLE seem to be due to its binding to opioid receptors; however, it inhibits the growth of LNCaP cells, devoid of opioid receptors. We have investigated the effects of DADLE on this cell line using transmission electron microscopy, immunocytochemistry and cytometry, in order to elucidate the general mechanism(s) by which this enkephalin affects cell metabolism. We demonstrated that, similar to cell lines provided with opioid receptors, in LNCaP cells DADLE induces structural modifications of cytoplasmic and nuclear constituents, as well as a decrease in transcription and proliferation. However, DADLE does not provoke an increase in apoptotic or necrotic cell fraction, and, after removing the enkephalin from the culture medium, all effects disappear. We also demonstrated that DADLE molecules enter the cytoplasm and the nucleus of LNCaP cells, mostly binding to perichromatin fibrils and dense fibrillar component, where transcription and early splicing of pre-mRNAs and pre-rRNAs occur. In conclusion, our data demonstrate that the effect of DADLE on transcription and on cultured cells does not depend on opioid receptors. DADLE can, therefore, be envisaged as an extremely promising molecule to be used for inducing a reversible hypometabolic state in various cultured cells, without provoking cell damage or death.

Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture

Cryoelectron microscopy of vitrified sections has become a powerful tool for investigating the fine structural features of cellular compartments. In the present study, this approach has been applied in order to explore the ultrastructural morphology of the interphase nucleus in different mammalian cultured cells. Rat hepatoma, Chinese hamster ovary and Potorus kidney cells were cryofixed by high-pressure freezing and the cryosections were examined at low temperature by transmission electron microscopy. Our results show that while the contrast of nuclear structural domains is remarkably homogeneous in hydrated sections, some of them can be recognised due to their characteristic texture. Thus, condensed chromatin appears finely granular and the perichromatin region contains rather abundant fibro-granular elements suggesting the presence of dispersed chromatin fibres and of perichromatin fibrils and granules. The interchromatin space looks homogeneous and interchromatin granules have not been identified under these preparative conditions. In the nucleolus, the most striking feature is the granular component, while the other parts of the nucleolar body, which appear less contrasted, are difficult to resolve. The nuclear envelope is easily recognisable with its regular perinuclear space and nuclear pore complexes. Our observations are discussed in the context of results obtained by other, more conventional electron microscopic methods.

Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

BackgroundThe Cajal body (CB) is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation.ResultsIn this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a.ConclusionTim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB.

Changes in extranucleolar transcription during actinomycin D-induced apoptosis.

Actinomycin D (AMD) inhibits DNA-dependent RNA polymerases and its selectivity depends on the concentration used; at very high concentrations it may also induce apoptosis. This study investigates the effects of different concentrations (0.01 to 1 microg/ml) of AMD on RNA transcription and maturation and on the organization of nuclear ribonucleoproteins (RNPs), and their relationship with apoptosis induction. Human HeLa cells were used as a model system. At the lowest concentration used, AMD induced the segregation of the nucleolar components and impaired r-RNA synthesis, as revealed by the decreased immunopositivity for bromo-uridine incorporation and for DNA/RNA hybrid molecules. The synthesis of pre-mRNAs, on the contrary, was active, while the immunolabeling of snRNP proteins and of the SC-35 splicing factor strongly decreased on perichromatin fibrils (where they are involved in co-transcriptional splicing). This suggests that the post-transcriptional maturation of extranucleolar RNAs was also affected. Moreover, still in the absence of typical late morphological or biochemical signs of apoptosis (i.e. chromatin condensation), these cells displayed the early apoptotic features, i.e. the externalization of phosphatidylserine residues on the plasma membrane and propidium iodide exclusion in vivo. At the highest concentrations of AMD used, apoptosis massively occurred, with the typical morphological events (progressive chromatin condensation, clustering of snRNPs and SC-35 splicing factor, cell blebbing). However, transcription of hnRNAs was maintained in the residual areas of diffuse chromatin up to advanced apoptotic stages. The inhibition of rRNA synthesis and the defective pre-mRNA maturation seem to be part of the apoptotic process induced by AMD.

Aging affects the distribution of the circadian CLOCK protein in rat hepatocytes

Several biochemical, physiological, and behavioral processes exhibit cyclic oscillations of about 24 h, which have been defined as circadian rhythms. In mammals, the primary circadian pacemaker resides in the suprachiasmatic nuclei; however, cell-autonomous circadian oscillators occur also in extraneural tissues, including the liver. CLOCK protein is a transcription factor essential for normal circadian rhythms and recent studies have demonstrated that it undergoes intranuclear redistribution in hepatocytes, along the daily cycle. It is known that aging leads to a progressive deterioration of the circadian rhythm at the behavioral, physiological, and cellular levels; in addition, aging affects the organization of nuclear structural components involved in transcription and splicing. In this view, we carried out ultrastructural immunocytochemical analyses on hepatocytes of adult and old rats, so as to investigate possible qualitative and quantitative modifications of CLOCK protein, in relation to the aging process. Our observations demonstrated that most CLOCK protein was always located in the cell nucleus, where it accumulated on perichromatin fibrils (the sites of premRNA transcription and early splicing); in addition, CLOCK showed daily oscillations in the different nuclear compartments, but these oscillations differed significantly between adult and old animals. This unusual distribution of CLOCK protein during aging could be related to the prolonged diurnal activity of old animals and/or to altered nuclear pathways.