Wilhelm bernhard workshop on the cell nucleus

Yuan L.. et al.: J. Cell Biol. 142:331-339, 1998

POSTERS

TRANSCRIPTION AND SPLICING IN THE TESTIS OF MICE FED ON A GENETICALLY MODIFIED-SOYBEAN DIET

Cisterna B.¹, Vecchio L.¹, Manuali E.², Malatesta M.³, Biggiogera M. <sup>1

1</sup>Dipartimento di Biologia Animale, Laboratorio di Biologia Cellulare, University of Pavia, ²Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, Perugia, and ³Istituto di Istologia ed Analisi di Laboratorio, University of Urbino “Carlo Bo”, Italy
We have focused our attention on some features of the transcription/splicing mechanism in the testis from mice fed on genetically modified (GM) soybean and sacrified after 1, 2, 5 and 8 months. Our data show a decrease in anti-RNA Polimerase II, in SC35 splicing factor and Sm labelling both in somatic cells (Sertoli cells) and in spermatogonia, spermatocytes and spermatids, after 1, 2 and 5 mounths on this diet. We have also observed an increase/accumulation of perichromatin granules (PG), containing mRNA, in the nucleus of Sertoli cells. Moreover, these cells display a significant decrease of nuclear pore. As for the morphology, in Sertoli cells (GM+), perichromatinic fibrils (PF) are numerous, nucleoli are more reticulated and larger, signal of high transcriptional activity. Moreover, many vescicles, probably correlated to SER are present in the cytoplasm. At 8 months, there is a resumption of transcription accompained by an increase in labelling for anti-RNA Pol II and anti-hnRNP. Considering that the increase of PG in GM-fed animals is probably due to a decrease of the nuclear pore density, some hypotheses can be proposed to explain this phenomenon, taking into account the fact that such an RNA accumulation seems to be transient.

actin is involved in the initiation of transcription by RNA polymerase II

Fuchsova B. ¹, Mavrommatis E.¹, Philimonenko V.V.³, Goodrich J.A.⁴, Hope T.J.², Lessard, J.L⁵, Hozak P.³ and de Lanerolle P.¹

Departments of ¹Physiology and Biophysics and ²Microbiology and Immunology, University of Illinois at Chicago, USA; ³Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic; ⁴Chemistry and Biochemistry, University of Colorado at Boulder, and ⁵Children's Hospital Research Foundation, Cincinnati, USA
Actin is an abundant protein in the cell nucleus and a number of studies have pointed to its role in transcription. We have demonstrated that  actin is present in RNA polymerase II (Pol II) preinitiation complexes (PIC) assembled on immobilized DNA template and co purifies with Pol II fraction from HeLa cell nuclei. In addition, we have shown that it co localizes with Pol II at EM level. Antibodies to  actin inhibit transcription by Pol II in vitro and when microinjected into the nuclei of living cells. To further characterize the role of  actin in PIC formation we studied its association with TATA-binding protein (TBP). Immunofluorescence studies showed that  actin co localizes with TBP in the nucleolus of HeLa cells but there was no significant overlap in the nucleoplasm. Introduction of new transcription sites by virus infection caused redistribution of  actin and TBP to the sites of intense transcription labeled by BrUTP incorporation. Moreover,  actin co localizes with the accumulations of TBP in the nucleus of adenovirus infected cells. The co localization of TBP and  actin was further confirmed at EM level. Taken together, these data suggest that  actin is involved in the initiation of transcription powered by Pol II.

modifications OF Transcription and splicing in ageing

Malatesta M.¹, Bertoni-Freddari C.², Fattoretti P.², Baldelli B.¹, Battistelli S.¹, and Biggiogera M.<sup>3

1</sup>Istituto di Istologia e Analisi di Laboratorio, University of Urbino “Carlo Bo”; ²Neurobiology of Aging Laboratory, INRCA Research Department, Ancona; ³Dipartimento di Biologia Animale, Laboratorio di Biologia Cellulare, University of Pavia, Italy
Ageing involves changes in transcription and splicing pathways. In a previous study we described an unusual accumulation of perichromatin fibrils (PFs) – the in situ form of pre-mRNA transcription and early splicing – in hepatocyte nuclei of old rats in comparison to adult animals. In this study we investigated, by means of immunoelectron microscopy, the nature of such PFs, analysing the presence of transcription and splicing factors and the incorporation of bromouridine (BrU) in adult (9 months) and old (28 months) rats. Our observations revealed that no difference in anti-snRNP and anti-fibrillarin labelling occurs between adult and old rats, whereas old animals showed lower signals for anti-polymerase II and anti-SC-35 probes. Anti-BrU immunolabelling, located on PFs and on the nucleolar dense fibrillar component, demonstrated a lower incorporation in the nucleoplasm in old rats, whereas no difference was found in the nucleolus. On the basis of our previous and present data, we can hypothesise altered mRNA pathways in hepatocyte nuclei of old rats. In fact, the lower BrU incorporation in the nucleoplasm together with the lower amounts of polymerase II in old rats indicate a decrease in pre-mRNA transcription rate. Moreover, the low amounts of nucleoplasmic splicing factors as well as the accumulation of perichromatin granules - storage and/or transport sites of spliced mRNA – previously observed in old rats suggest also a slow down of PF processing and/or transport in ageing. Such phenomenon would explain the persistence in the nucleoplasm of pre-mRNA and/or mRNA as PFs in old animals. As for the nucleolus, our data indicate no modification in pre-rRNA transcription and early splicing in old rats. During ageing a general reduction in protein and RNA synthesis occurs in liver. It is possible that these changes would be related to the alterations in both transcription and processing of pre-mRNA.

THE PLANT NUCLEOLIN-LIKE PROTEIN NopA100 IS PROTEOLYZED AND RE-ORGANIZED IN RELATION TO THE NUCLEOLAR ACTIVITY AND CELL CYCLE PROGRESSION

González-Camacho F., Medina F.J.

Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
The nucleolar protein NopA100 has been detected in the soluble nuclear fraction from onion proliferating cells. From its physico-chemical properties, and from its immunological cross-reactions, it can be identified as a nucleolin-like protein. In a cell population of root meristems synchronized with hydroxyurea the levels of the protein were shown to increase in the G2 phase of cell cycle. Light and electron microscopical immuno-cytochemistry revealed an unequal distribution within the nucleolus appearing mainly in the dense fibrillar component, near fibrillar centers. A Western blot analysis with anti-NopA100 antibody resulted in the intense labeling of a band of 100 kDa, but also of a series of proteins related with it, one of them higher in molecular mass (120 kDa) and several of them below this level, suggesting that NopA100 undergoes a physiological process of proteolytic maturation, similar to that described for mammalian nucleolin, but not reported for other nucleolin-like proteins from Xenopus, other plants and yeasts. There is a relationship between the increase of the activity of nucleolus, the activity of NopA100, the progress of the cell cycle and NopA100 proteolysis. This relationship has been evidenced after a treatment with leupeptin, a general inhibitor of proteases, of nuclei extracted from synchronized cells, which resulted in the differential quantitative diminution of the bands product of the proteolysis at the expenses of an increase of the 100 kDa main band, according to cell cycle stages. The variations described in different features of NopA100 during the cell cycle are accompanied by a structural rearrangement of the nucleolus, which is expressed in quantitative changes of the relative contribution of the different nucleolar subcomponents to the nucleolar volume. A structural model of nucleolar organization can be designed for each one of the periods of the interphase in onion proliferating cells.


ISOLATION AND CHARACTERIZATION OF TOPOGENIC PROTEIN COMPLEXES PRESENT IN THE NUCLEOLUS

Hanisch C. and Schmidt-Zachmann M. S.

German Cancer Research Center, Division of Cell Biology, Heidelberg, Germany
The nucleolus which represents an accumulation of rDNA and its transcription products together with a characteristic set of proteins, is a complex nuclear substructure in which key steps of ribosome biogenesis take place. More recently, it turned out that the nucleolus is also involved in several other cellular processes such as cell cycle control, aging and mRNA export. Over the years we have reported on the identification of several constitutive nucleolar proteins (e.g. proteins NO29, NO38, NOH61), which have different roles in the complex process of ribosome biogenesis. One major aim of our recent studies will be the identification of other nucleolar proteins interacting with these components, e.g. i) by co-immuno-precipitation using the appropriate protein-specific antibodies, ii) using the tandem affinity purification (TAP) method, a technique that combines two high-affinity steps under native conditions and iii) by the yeast two-hybrid system, which in particular allows the identification of transient interaction partners. Our lab has reported the discovery and molecular characterization of the very acidic nucleolar protein, termed NO29. This protein is sequence-related to the histone-binding protein nucleoplasmin and to the major nucleolar protein NO38, i.e. it represents an additional member of the "nucleoplasmin family" (Zirwes et al. 1997, Proc. Natl. Acad. Sci. USA 94, 11387-92). Recently, we have generated a panel of mono- as well as polyclonal antibodies specifically reacting with protein NO29, which we used for immunoprecipitation experiments. Upon SDS-PAGE, a number of co-precipitating polypeptides became visible, which will be analyzed by MALDI-TOF mass spectrometry. Finally, we started to elucidate the functional roles of these non-ribosomal nucleolar proteins and their importance in maintaining the specific structures by RNA interference (RNAi).


FUNCTIONAL ORGANIZATION OF THE POLYTENE CELL NUCLEOLUS

Rodríguez-Vilariño, V; Díez, J.L.; Medina, F.J.

Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
In polytene cells, DNA is amplified forming the polytene or giant chromosomes, as a result of lateral amplification of the homologous chromatids that remain associated throughout successive rounds of replication. Ribosomal genes result, therefore, amplified, and their activity gives rise to the formation of a giant nucleolus, which may be called “Polytene Cell Nucleolus” (PCN). We have studied some basic aspects of the organization of the PCN using as a model the polytene cells of salivary glands from Chironomus larvae, characterized by a high level of polyteny, and a nucleolus measuring as much as 10-15 µm in diameter. The organization of nucleolar subcomponents in these cells has been defined at the ultrastructural level, being composed of the dense fibrillar component (DFC) and the granular component (GC); the fact that fibrillar centers (FC) have never been identified in these nucleoli, either under physiological conditions or after the use of inhibitors, re-opens the earlier debate, probably closed with insufficient evidence, on the functional significance of these structures in the nucleolar activity. In general, modifications and rearrangements of nucleolar subcomponents have been studied in relation to the nucleolar activity. By means of immunocytochemistry and confocal microscopy we have studied the organization of DNA within the PCN. The results showed that this organization was highly dynamic, as indicated by drastic changes in the spatial distribution, associated to physiological changes. The use of FISH has made possible the definition of the location of rDNA in the context of total nucleolar DNA and its structural changes associated to the activity of the nucleolus. Under physiological conditions, no rDNA has been located in condensed chromosomal bands; on the contrary, different levels of decondensation have been appreciated, until the total extension of the innermost transcribing (or ready for transcription) segments. On the other hand, the immunodetection of fibrillarin and nucleolin, two nucleolar proteins involved in pre-rRNA processing, has been carried out, allowing us to differentiate specific subnucleolar domains. In general, the organization of the PCN suggests that it functions as a single entity, and not as the juxtaposition of the activity of multiple NORs. Finally, we have explored the possibilities of the nucleolus for the study of the location of proteins not related with ribosome biogenesis, giving account of the so-called “plurifunctional nucleolus”.


THE NUCLEOLUS OF THE TELEOST FISH Barbus barbus (L): AN EXAMPLE OF NUCLEOLUS WITHOUT FIBRILLAR CENTER

Thiry M.

Laboratoire de biologie cellulaire, Département des Sciences de la vie, Université de Liège, Institut A. Swaen, Liège, Belgium
Nucleoli are ubiquitous structural elements of eucaryotic interphase cell nuclei. They are the morphological structures where rRNA genes are expressed. On the basis of their structural compartmentalization, two types of nucleoli can be distinguished. The first type of nucleoli contains three main nucleolar components: the fibrillar centers (FCs), the dense fibrillar component (DFC), and the granular component (GC). It is typically accounted in mammalian cells. In the second type of nucleoli, in addition to a GC it contains only one fibrillar component. This ultrastructural feature is characteristic of amphibian oocytes where it is well known, but also seems to occur in somatic and germinal cells of many invertebrate and anamniote vertebrate species. In the present study, we studied the evolution of the nucleoli during oogenesis in the teleost fish Barbus barbus (L) using light and electron microscopies. We distinguished eight successive stages in nucleolar structure : (I) a single, compact, central nucleolus in the lobed nucleus of the oogonium, (II) the nucleolus remained compact, it lay in the central area of the oval nucleus of the leptotene oocyte, (III) the nucleolus became peripheral in the zygotene oocyte, (IV) a few tiny nucleoli appearing in the DNA cap of the pachytene oocyte, (V) several enlarged and vacuolized nucleoli at the beginning of the diplotene stage, (VI) Fragmentation of nucleoli into multiple spherical nucleoli with a fibrillar core and several short granular arches at their surface during the end of previtellogenesis, (VII) multiple reticulated nucleoli with a fibrillar core surrounded by a well-developped network of granular and fibrillar ribbons in the early vitellogenic oocyte, (VIII) Nucleoli fragmented into fibrillar and granular remains in the late vitellogenic oocyte. By cytochemical and immunocytological methods, we showed further that the nucleoli of germ and follicle cells comprised a fibrillar, silver-stainable component and a granular component whatever the oogenetic stage. Using the TdT method, we detected DNA, a characteristic constituent of fibrillar centers in the mammalian cell nucleolus, in the fibrillar component of the fish nucleolus. The nucleolar protein, fibrillarin, a marker of the dense fibrillar component in the mammalian cell nucleolus, was also located in the single fibrillar part of the Barbus nucleolus. These data supports the view that the nucleolus of the teleost fish, Barbus barbus, is an excellent model of nucleolus without fibrillar center.