Clusters Of Granules Research Articles

Nuclear speckles.

Nuclear speckles, also known as interchromatin granule clusters, are nuclear domains enriched in pre-mRNA splicing factors, located in the interchromatin regions of the nucleoplasm of mammalian cells. When observed by immunofluorescence microscopy, they usually appear as 20-50 irregularly shaped structures that vary in size. Speckles are dynamic structures, and their constituents can exchange continuously with the nucleoplasm and other nuclear locations, including active transcription sites. Studies on the composition, structure, and dynamics of speckles have provided an important paradigm for understanding the functional organization of the nucleus and the dynamics of the gene expression machinery.

Ediacaran ice-rafting and coeval asteroid impact, South Australia: insights into the terminal Proterozoic environment

Isolated quartzose pebbles, clusters of quartz granules, orthogonal aggregates of poorly sorted quartzose coarse sand, and ovoid pellets (≤2 mm long) of quartz silt occur in hemipelagic marine mudstone of the mid-Ediacaran Bunyeroo Formation exposed in the Adelaide Geosyncline (Adelaide Rift Complex), and ovoid pellets of quartz silt in cores of the correlative marine Dey Dey Mudstone from deep drillholes in the Officer Basin, South Australia. This detritus is interpreted respectively as dropstones, dumps, and frozen aggregates dispersed by sea ice possibly of seasonal origin, and till pellets transported by glacial ice. The ice-rafted material in the Bunyeroo Formation only has been found <10 m stratigraphically below and above a horizon of dacitic ejecta related to the 90 km diameter Acraman impact structure in the Mesoproterozoic Gawler Range Volcanics 300 km to the west. Furthermore, till pellets have been identified 4.4 to 68 m below distal Acraman ejecta in the Dey Dey Mudstone >500 km northwest of the impact site. The Acraman impact took place at a low paleolatitude (∼12.5°) and would have adversely affected the global environment. The stratigraphic observations imply, however, that the impact occurred during, but did not trigger, a cold interval marked by sea ice and glacial ice, although the temporal relationship with Ediacaran glaciations elsewhere in Australia and on other continents is unclear. Release from the combined environmental stresses of a frigid, glacial climate near sea-level and a major impact in low latitudes may have been a factor influencing subsequent Ediacaran biotic evolution.

Nuclear Organization and Dynamics of 7SK RNA in Regulating Gene Expression

Noncoding RNAs play important roles in various aspects of gene regulation. We have identified 7SK RNA to be enriched in nuclear speckles or interchromatin granule clusters (IGCs), a subnuclear domain enriched in pre-mRNA processing factors. 7SK RNA, in association with HEXIM 1 and 2, is involved in the inhibition of transcriptional elongation by RNA polymerase II. Inhibition occurs via sequestration of the active P-TEFb kinase complex (CDK 9 and Cyclin T1/T2a/b or K) that is involved in phosphorylating the C-terminal domain of RNA polymerase II. Our results demonstrate that knock-down of 7SK RNA, by specific antisense oligonucleotides, results in the mislocalization of nuclear speckle constituents in a transcription-dependent manner, and the transcriptional up-regulation of a RNA polymerase II transcribed reporter gene locus. Furthermore, 7SK RNA transiently associates with a stably integrated reporter gene locus upon transcriptional down-regulation and its presence correlates with the efficient displacement of P-TEFb constituents from the locus. Our results suggest that 7SK RNA plays a role in modulating the available level of P-TEFb upon transcriptional down-regulation by sequestering its constituents in nuclear speckles.

A new species of the Lower Ordovician pliomerid trilobite Pseudocybele and its biostratigraphic significance

A low diversity trilobite fauna consisting entirely of new species occurs at several horizons in the Blackhillsian Stage (Floian), in a narrow stratigraphic interval high in the Fillmore Formation in the southern Confusion Range, Ibex area, Millard County, western Utah. This is the type section of the Blackhillsian Stage, the highest of the four stages with comprise the Ibexian Series, the Laurentian Lower Ordovician. The interval occurs between the underlying Presbynileus ibexensis Zone and the overlying "Pseudocybele nasuta Zone". Previous studies assigned the interval to the P. nasuta Zone, but new collections show that the fauna is unique and shares no species with either the underlying or overlying assemblages. It was recognized in 2009 as the Pseudocybele paranasuta Zone, but the name bearer was not formally described. Pseudocybele paranasuta n. sp. is a distinctive pliomerid trilobite with diagnostic features including: clusters of granules on the frontal lobe of the glabella and the middle body of the hypostome; maculae along the lateral branches of the middle furrow of the hypostome; a short, slender median hypostomal spine; and a strong, W-shaped impression in the terminal piece of the pygidium.

Amylose involvement in the amylopectin clusters of potato starch granules

The granular organization of common corn and potato starch is characteristically different. The objective of this study was to investigate iodine complex formation with starch lintners from common corn starch (CCS) and potato starch (PS) litners as a function of water content. Starches were subjected to mild acid hydrolysis (lintnerization). Size exclusion chromatography of the lintners indicated that the linear chains remaining after lintnerization had smaller degree of polymerization in PS lintners than in the corresponding CCS lintners. For both CCS and PS, the absorbance intensity and the wavelength of maximum absorption ( λ max) of molecularly dispersed starches, in diluted iodine solution, decreased with increasing lintnerization extent. When the granular lintners were exposed to iodine vapor, following equilibration to different moisture contents, the reduction in the iodine binding was evident in PS lintners but not in CCS lintners. Furthermore, the iodination partially destroyed the crystallinity of native PS granules but not that of CCS granules. However, B-type crystallinity was still evident in PS lintners. This behavior was attributed to different location of amylose within starch granules, supporting the involvement of amylose in the B-type crystallites of PS, and the independence of amylose from the A-type crystallites of CCS.

Mouse Dfa Is a Repressor of TATA-box Promoters and Interacts with the Abt1 Activator of Basal Transcription

Our study of the mouse Ate1 arginyltransferase, a component of the N-end rule pathway, has shown that Ate1 pre-mRNA is produced from a bidirectional promoter that also expresses, in the opposite direction, a previously uncharacterized gene (Hu, R. G., Brower, C. S., Wang, H., Davydov, I. V., Sheng, J., Zhou, J., Kwon, Y. T., and Varshavsky, A. (2006) J. Biol. Chem. 281, 32559-32573). In this work, we began analyzing this gene, termed Dfa (divergent from Ate1). Mouse Dfa was found to be transcribed from both the bidirectional P(Ate1/Dfa) promoter and other nearby promoters. The resulting transcripts are alternatively spliced, yielding a complex set of Dfa mRNAs that are present largely, although not exclusively, in the testis. A specific Dfa mRNA encodes, via its 3'-terminal exon, a 217-residue protein termed Dfa(A). Other Dfa mRNAs also contain this exon. Dfa(A) is sequelogous (similar in sequence) to a region of the human/mouse HTEX4 protein, whose physiological function is unknown. We produced an affinity-purified antibody to recombinant mouse Dfa(A) that detected a 35-kDa protein in the mouse testis and in several cell lines. Experiments in which RNA interference was used to down-regulate Dfa indicated that the 35-kDa protein was indeed Dfa(A). Furthermore, Dfa(A) was present in the interchromatin granule clusters and was also found to bind to the Ggnbp1 gametogenetin-binding protein-1 and to the Abt1 activator of basal transcription that interacts with the TATA-binding protein. Given these results, RNA interference was used to probe the influence of Dfa levels in luciferase reporter assays. We found that Dfa(A) acts as a repressor of TATA-box transcriptional promoters.

Interchromatin granule clusters of mouse preovulatory oocytes: Organization, molecular composition, and possible functions

At the diplotene stage of meiotic prophase, the nucleus of mouse preovulatory oocytes contains multiple interchromatin granule clusters (IGC). These nuclear compartments are universal and evolutionary conserved and enriched in pre-mRNA splicing factors. Nowadays, IGCs are believed to play an important role in gene expression events and contain different molecular components that allow coupling of many processes from transcription to mRNA export. We obtained the data on the distributions of poly(A)+RNA, hnRNPS A/B, and NXF1/TAP factor of mRNA export. These factors were found to associate with IGCs of mouse preovulatory oocytes. In the present study, we have demonstrated for the first time the dynamics of large IGCs after specific phosphorilation of SR-proteins with okadaic acid, an inhibitor of protein phosphatases. Using electron microscopy, conventional fluorescent and confocal microscopies, as well as microinjections of olygonucleotide probes in mouse oocytes, some features of structural organization and molecular compositions of IGCs in the nuclei of mouse oocyte from antral follicles were established. Possible roles of IGCs in pre-mRNA metabolism and the participation of these structures in mRNA export are discussed.

Aggregation induced polyelectrolyte multilayer film containing cubic silsesquioxane nanoparticles

Polyelectrolyte multilayer (PEM) films offer a method to functionalize substrates with specific properties that enable the films to be used for a variety of purposes. Desirable qualities of PEM films can include mechanical strengths, ease of preparations, flexibility, and their abilities to have their properties tailored to suit a particular process. We present a simple method to fabricate a class of PEM films that incorporate cubic silsesquioxane nanoparticles (CSSQ). Through a spin self-assembly (SSA) process, a hybrid multilayered film with two-components, namely, poly(styrene sulfonate) (PSS) and octaammonium cubic silsesquioxane (CSSQ) nanoparticle have been fabricated. The formation of this multilayer film is further verified by ellipsometry, contact angle studies, and atomic force microscopy (AFM). The water contact angle and ellipsometric measurements exhibit that the (PSS/OA-CSSQ) films are deposited onto the substrate. The surface topography of the deposited bilayers of PSS/OA-CSSQ film appears to be uniformly distributed with extremely small granules but the film uniformity of the granular surface is diminished and clusters of granules are observed at above 5 bilayers due to the aggregation of the OA-CSSQ nanoparticles.

352 DISSOCIATION OF NUCLEAR AND CYTOPLASMIC MATURATION OF BOVINE OOCYTES IN DEFINED CULTURE SYSTEM

Insufficient cytoplasmic maturation of MII oocytes may be one reason of the low rate of embryo production in vitro. To solve this problem, a two-step procedure of IVM is tested. In this case, there is a prematuration period (PMP) in which meiosis is arrested, followed by a resumption period (RP) during which the oocytes are liberated from the inhibition of meiosis. Preliminary studies have shown that a defined medium (DM) inhibits nuclear oocyte maturation (NM) (Unpublished data). Thus, our purpose was to verify if meiosis arrest is followed by an inhibition of cytoplasmic maturation (CM). Herein we investigated the effects of DM on NM and CM using the two-step procedure for IVM. COCs were selected from bovine ovaries and cultured in non-defined medium (NDM), composed of TCM-199 suplemented with 10% fetal calf serum and 200 ηg mL-1 FSH or in a defined medium (DM), MEMα supplemented with 0.1% Polyvinyl Alcohol. Both media are from Invitrogen-Gibco/BRL, Grand Island, NY, USA. In the PMP (experiment 1), COCs (n = 232) were distributed in G1 (NDM) or G2 (DM) and cultured for 24 h as usual. After this, NM and CM were analyzed. As inhibition of meiosis was observed in G2, a second step of culture (experiment 2) was performed to evaluate the ability of the COCs to reverse meiosis arrest. Thus, COCs (n =202) were distributed in G1 (NDM) or G3 (DM +NDM). COCs from G1 were cultivated in NDM for 24 h, and that ones from G3 in DM for 24 h followed by cultivation in NDM for an additional 24 h (RP). After this, NM and CM were analyzed. The CM evaluation (distribution of organelles) was performed in fresh oocytes (0 h) and in G1, G2, and G3 after usual preparation for transmission electron microscopy (TEM) analysis. The NM data were analyzed by Chi-square test. After the PMP period, G1 COCs presented 83.17% (84/101) of complete nuclear maturation whereas G2 presented 74.05% (97/131) of NM inhibition (oocytes in GV or GVBD) (P &lt; 0.05). This meiosis arrest observed in DM was reversed after 24 h of cultivation in NDM, 79.17% (90/110) of G3 COCs were at MII. The analysis of CM has shown fresh COCs with ultrastructural characteristics of immaturity, as presence of mitochondria in the periphery of the ooplasm, and cortical granule clusters in the ooplasm; G1 COCs with mature cytoplasmic characteristics as mitochondria spread throughout the ooplasm and cortical granules located close to the oolema and G2 COCs with both immature and mature characteristics as presence of ooplasm granule clusters and presence of mitochondria spread throughout the ooplasm. G3 have shown mature cytoplasmic characteristics in the end of RP. In conclusion, a two-step procedure of IVM using a DM was able to maintain meiosis arrest while the cytoplasm undergoes maturation (G2). This could be a strategy to improve the rate of viable embryos obtained at in vitro embryo production. FAP-DF, Finatec, CNPq, Capes.

Role of epidermal growth factor receptor (EGFR) in corneal remodelling in diabetes

This study examined the role of epidermal growth factor receptor (EGFR) signalling on the organization and remodelling of collagen fibrils (CFs) and proteoglycans (PGs) in the stroma of diabetic rat cornea. Diabetes was induced in female Wistar rats (n = 5) by streptozotocin (STZ) injection (55 mg/kg). Treatment with a selective inhibitor of EGFR tyrosine kinase, AG1478, was started on the same day as the induction of diabetes and administered every other day for 4 weeks. Corneas were fixed in 4% paraformaldehyde at 4 degrees to allow for analysis of CF diameters and in 2.5% glutaraldehyde in sodium acetate buffer containing cuprolinic blue to enable the study of PG distribution. AnalySIS soft imaging software was used to analyse CFs and PGs. Epithelial thickness, and median diameter and area fraction of CF in corneal stroma were decreased in diabetic rat cornea compared with normal cornea (p < 0.001), whereas the median PG area and area fractions were significantly increased (p < 0.001). Treatment with AG1478, although it had no action on normal cornea, prevented these diameter and area fraction changes in CFs and PGs. The cornea of AG1478-treated diabetic rats showed a slight increase in CF diameter and area fraction and a decreased number density. These data show that the distribution of corneal stroma CFs and PGs was altered after 4 weeks of diabetes and that, furthermore, treatment with an EGFR signalling inhibitor normalized these abnormalities. The data suggest that EGFR plays an important role in the development of diabetes-induced corneal remodelling.

Localization of poly(A)+ RNA and mRNA export factors in interchromatin granule clusters of two-cell mouse embryos

Interchromatin granule clusters (IGCs), also known as nuclear speckles, splicing factor compartments, or SC35-domains, are one of the most universal nuclear organelles of the cell. We have used two-cell mouse embryos as an experimental system to study the possible association of poly(A)(+) RNA and factors involved in RNA export (Tip-associated protein [TAP] and heterogenous nuclear ribonucleoprotein A/B [hnRNP A/B]) with IGCs. Poly(A)(+) RNA was localized by microinjections of 2'-O-Me(U)(22) probes conjugated with tetramethylrhodamine. RNA export proteins were detected by immunofluorescence confocal laser microscopy and immunogold-labeling electron microscopy. We found that poly(A)(+) RNA was located in IGCs regardless of the transcriptional state of the nuclei. hnRNP A/B was also found to characterize IGCs of the embryo nuclei with various levels of transcription activity. In transcriptionally active embryo nuclei, TAP was detected in the vicinity of IGCs rather than inside them; however, when transcription was inhibited by drugs, TAP was localized to IGCs. Our data support the idea that IGCs not only serve as splicing factor reservoirs, but also take part in mRNA retention and export.

Distinct domanial and lamellar distribution of clustered lipofuscin granules in microglia in the main olfactory bulb of young mice

Lipofuscin granules are generally considered as age-pigment. However, we encountered numerous large irregular clusters of lipofuscin granules in the olfactory nerve layer and glomerular layer of the main olfactory bulb (MOB) of young adult and even juvenile mice of C57BL/6J strain. Those numerous autofluorescent irregular lipofuscin granules were contained in the cytoplasm of microglial cells. Importantly they showed a prominent pattern of distribution; that is, they were rather restricted to the OCAM positive ventro-lateral domain (V-domain) of the MOB but few in the OCAM negative dorso-medial domain (D-domain), even when microglia distributed rather homogeneously in both OCAM positive V-domain and OCAM negative D-domain. Those lipofuscin granules were not seen in MOBs of 10 days and 2w old C57BL mice, but usually encountered in the MOBs of 3w old mice. Similar clusters of lipofuscin granules in the olfactory nerve layer and glomerular layer were also encountered in BALB/c strain, and, although less prominent, in ICR and ddY strains. However, they were not encountered in young adult rats of three strains, Wistar, Sprague–Dawley and Long-Evans, indicating one of prominent species differences between mice and rats.

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.

Factors related to RNA polymerase II transcription are localized in interchromatin granule clusters of Panorpa communis oocytes.

Diplotene oocyte nucleus of the scorpionfly Panorpa communis is transcriptionally silent and contains numerous nuclear bodies including interchromatin granule clusters (IGCs). The latter consist of the granules of 30-50 nm in diameter and contain IGC marker protein SC35 as well as RNA polymerase II. In this study, we also localized in P. communis oocyte IGCs the transcription coactivators CBP/p300, TATA-binding protein (TBP) which is a component of the basal transcription factor TFIID and the basal transcription factor TFIIH. We belive that IGCs in transcriptionally inert P. communis oocytes are storage sites for the components of RNA polymerase II holoenzyme and other factors of RNA pol II transcription.

TDP-43 in ubiquitinated inclusions in the inferior olives in frontotemporal lobar degeneration and in other neurodegenerative diseases: a degenerative process distinct from normal ageing

Ubiquitin immunoreactive (UBQ-ir) inclusions were present to variable extents in the inferior olivary nucleus (ION) in 37/48 (77%) patients with frontotemporal lobar degeneration (FTLD), in 10/11 (91%) patients with motor neurone disease (MND), in 5/5 (100%) patients with Alzheimer's disease (AD), 5/7 (71%) patients with dementia with Lewy bodies, 13/19 (68%) patients with Parkinson's disease, 11/11(100%) patients with Progressive Supranuclear Palsy, 2/6 (33%) patients with Multisystem Atrophy, 1/3 (33%) patients with Huntington's disease and in 14/14 (100%) normal elderly control subjects. In FTLD, UBQ-ir inclusions were present in 26/32 (81%) patients with FTLD-U, in 10/15 (67%) patients with tauopathy, and in the single patient with Dementia Lacking Distinctive Histology. In 13 FTLD-U patients, and in a single AD and in 2 MND patients, the UBQ-ir inclusions had a rounded, spicular or skein-type appearance, and these were also TDP-43 immunoreactive (TDP-43-ir). In all other affected patients in all diagnostic groups, and in control subjects, the UBQ-ir neuronal cytoplasmic inclusions (NCI) were of a conglomerated type, resembling a cluster of large granules or globules, but were never TDP-43-ir. In 3 of the 13 FTLD-U patients with spicular NCI, conglomerated NCI were also present but in separate cells. Double-labelling immunohistochemistry, and confocal microscopy, for UBQ and TDP-43 confirmed that only the spicular UBQ-ir inclusions in patients with FTLD-U, AD and MND contained TDP-43, though in these patients there were occasional TDP-43 immunoreactive inclusions that were not UBQ-ir. Nuclear TDP-43 immunoreactivity was absent in ION in FTLD-U, AD or MND when TDP-43 cytoplasmic inclusions were present, but remained in neurones with UBQ-ir, TDP-43 negative inclusions. The target protein within the UBQ-ir, TDP-43-negative inclusions remains unknown, but present studies indicate that this is not tau, neurofilament or internexin proteins. These TDP-43 negative, UBQ-ir inclusions appear to be more related to ageing than neurodegeneration, and are without apparent diagnostic significance. The pathophysiological mechanism leading to their formation, and any consequences their presence may have on nerve cell function, remain unknown.

Universal nuclear domains of somatic and germ cells: some lessons from oocyte interchromatin granule cluster and Cajal body structure and molecular composition

It is now clear that two prominent nuclear domains, interchromatin granule clusters (IGCs) and Cajal bodies (CBs), contribute to the highly ordered organization of the extrachromosomal space of the cell nucleus. These functional domains represent structurally stable but highly dynamic nuclear organelles enriched in factors that are required for different nuclear activities, especially RNA biogenesis. IGCs are considered to be the main sites for storage, assembly, and/or recycling of the essential spliceosome components. CBs are involved in the biogenesis of several classes of small RNPs as well as the modification of newly assembled small nuclear RNA. We have summarized data on the molecular composition, structure, and functional roles of IGCs and CBs in the nuclei of mammalian somatic cells and oocytes of some animals with a special focus on insects. We have focused on similarities and differences between the IGCs and CBs of oocytes and the well-studied CBs and IGCs of cultured mammalian somatic cells. We have shown the heterogeneous character of oocyte IGCs and CBs, both in structure and molecular content. We have also demonstrated the unique capacity of oocytes to form close structural interactions between IGC and CB components. We proposed to consider these joint structures as integrated entities, sharing the features of both IGCs and CBs.

Towards a clinico-pathological classification of granule cell dispersion in human mesial temporal lobe epilepsies

The dentate gyrus (DG) plays a pivotal role in the functional and anatomical organization of the hippocampus and is involved in learning and memory formation. However, the impact of structural DG abnormalities, i.e., granule cell dispersion (GCD), for hippocampal seizure susceptibility and its association with distinct lesion patterns in epileptic disorders, such as mesial temporal sclerosis (MTS) remains enigmatic and a large spectrum of pathological changes has been recognized. Here, we propose a clinico-pathological classification of DG pathology based on the examination of 96 surgically resected hippocampal specimens obtained from patients with chronic temporal lobe epilepsy (TLE). We observed three different histological patterns. (1) A normal granule cell layer was identified in 11 patients (no-GCP; 18.7%). (2) Substantial granule cell loss was evident in 36 patients (referred to as granule cell pathology (GCP) Type 1; 37.5%). (3) Architectural abnormalities were observed in 49 specimens, including one or more of the following features: granule cell dispersion, ectopic neurons or clusters of neurons in the molecular layer, or bi-lamination (GCP Type 2; 51%). Cell loss was always encountered in this latter cohort. Seventy-eight patients of our present series suffered from MTS (81.3%). Intriguingly, all MTS patients displayed a compromised DG, 31 (40%) with significant cell loss (Type 1) and 47 (60%) with GCD (Type 2). In 18 patients without MTS (18.7%), seven displayed focally restricted DG abnormalities, either cell loss (n = 5) or GCD (n = 2). Clinical histories revealed a significant association between DG pathology patterns and higher age at epilepsy surgery (p = 0.008), longer epilepsy duration (p = 0.004), but also with learning dysfunction (p < 0.05). There was no correlation with the extent of pyramidal cell loss in adjacent hippocampal segments nor with postsurgical seizure relief. The association with long-term seizure histories and cognitive dysfunction is remarkable and may point to a compromised regenerative capacity of the DG in this cohort of TLE patients.

Cancer/testis antigen 45 is expressed in a nuclear speckles-like pattern in human tumor cell lines

The cancer/testis antigen (CT) family is defined by its specific expression pattern. In most cases CT antigens are expressed in normal human tissue only in germline cells and some tumors. Because of their restricted expression pattern CT antigens are regarded as potential targets for vaccine immunotherapy. Little is known about the functions of the various CT antigens. Some of these CT antigens (CT 7; MAGE A3/6) seem to be involved in the dysregulation of cell-cycle control and increased cell proliferation. The monoclonal antibody Ki-A10 detects a nuclear antigen with a unique distribution pattern in normal human tissues and tumors. The antigen is now characterized as cancer/testis antigen 45 (CT45). The accurate localisation of CT45 could provide details about its function. Therefore immunfluorescence stainings with subsequent confocal laser microscope analysis were performed to deliver precise data about the nuclear localisation of CT45. Different human tumor celllines (L428, HT1080, WS1-CLS, SW872) and 5-aza-2'-deoxycytidine treated human peripheral blood lymphocytes were stained with mab Ki-A10. All stainings showed an irregular and dotlike pattern, which is wellknown for nuclear speckles (interchromatin granule clusters). Nuclear speckles are dynamic structures known to harbour many proteins of the pre-mRNA splicing machinery. In addition CT45 shows a clear homology to a DEAD-Box protein (DDX26). DEAD-Box proteins are generally believed to be RNA helicases, which in turn are localised in nuclear speckles. Due to these results we believe that CT45 could be involved in pre-mRNA splicing.

Evidence for Insertional RNA Editing in Humans

Large-scale analysis directly at the protein level holds the promise of uncovering features not apparent or present at the gene level [1-3]. Although mass spectrometry (MS)-based proteomics can now identify and quantify thousands of cellular proteins in large-scale proteomics experiments, much of the peptide information contained in these experiments remains unassigned [4]. Here, we use such information to discover a previously unreported mechanism creating altered protein forms. Linker histones H1 and high-mobility group (HMG) proteins are abundant nuclear proteins that regulate gene expression through modulation of chromatin structure [5-8]. In the high-resolution MS analysis of histone H1 and HMG protein fractions isolated from human cells, we discovered peptides that mapped upstream of the known translation start sites of these genes. No alternative upstream start site exists in the genome, but analysis of Expressed Sequence Tag (EST) databases revealed that these N-terminally extended (ET) proteins are due to in-frame translation of the 5' untranslated region (5'UTR) sequences of the transcripts. The new translation start sites are created by a single uridine insertion between AG, reflecting a previously unreported RNA-editing mechanism. To our knowledge, this is the first report of RNA-insertion editing in humans and may be an example of the type of discoveries possible with modern proteomics methods.

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.