Functional analysis of adenovirus protein IX identifies domains involved in capsid stability, transcriptional activity, and nuclear reorganization.

The product of adenovirus (Ad) type 5 gene IX (pIX) is known to actively participate in the stability of the viral icosahedron, acting as a capsid cement. We have previously demonstrated that pIX is also a transcriptional activator of several viral and cellular TATA-containing promoters, likely contributing to the transactivation of the Ad expression program. By extensive mutagenesis, we have now delineated the functional domains involved in each of the pIX properties: residues 22 to 26 of the highly conserved N-terminal domain are crucial for incorporation of the protein into the virion; specific residues of the C-terminal leucine repeat are responsible for pIX interactions with itself and possibly other proteins, a property that is critical for pIX transcriptional activity. We also show that pIX takes part in the virus-induced nuclear reorganization of late infected cells: the protein induces, most likely through self-assembly, the formation of specific nuclear structures which appear as dispersed nuclear globules by immunofluorescence staining and as clear amorphous spherical inclusions by electron microscopy. The integrity of the leucine repeat appears to be essential for the formation and nuclear retention of these inclusions. Together, our results demonstrate the multifunctional nature of pIX and provide new insights into Ad biology.

The cell-surface-expressed nucleolin is associated with the actin cytoskeleton.

Nucleolin is a RNA- and protein-binding multifunctional protein. Mainly characterized as a nucleolar protein, nucleolin is continuously expressed on the surface of different types of cells along with its intracellular pool within the nucleus and cytoplasm. By confocal and electron microscopy using specific antibodies against nucleolin, we show that cytoplasmic nucleolin is found in small vesicles that appear to translocate nucleolin to the cell surface. Translocation of nucleolin is markedly reduced at low temperature or in serum-free medium, whereas conventional inhibitors of intracellular glycoprotein transport have no effect. Thus, translocation of nucleolin is the consequence of an active transport by a pathway which is independent of the endoplasmic reticulum-Golgi complex. The cell-surface-expressed nucleolin becomes clustered at the external side of the plasma membrane when cross-linked by the nucleolin-specific monoclonal antibody mAb D3. This clustering, occurring at 20 degrees C and in a well-organized pattern, is dependent on the existence of an intact actin cytoskeleton. At 37 degrees C, mAb D3 becomes internalized, thus illustrating that surface nucleolin can mediate intracellular import of specific ligands. Our results point out that nucleolin should also be considered a component of the cell surface where it could be functional as a cell surface receptor for various ligands reported before.

DNA replication progresses on the periphery of nuclear aggregates formed by the BCL6 transcription factor.

The BCL6 proto-oncogene, frequently alterated in non-Hodgkin lymphoma, encodes a POZ/zinc finger protein that localizes into discrete nuclear subdomains. Upon prolonged BCL6 overexpression in cells bearing an inducible BCL6 allele (UTA-L cells), these subdomains apparently coincide with sites of DNA synthesis. Here, we explore the relationship between BCL6 and replication by both electron and confocal laser scanning microscopy. First, by electron microscope analyses, we found that endogenous BCL6 is associated with replication foci. Moreover, we show that a relatively low expression level of BCL6 reached after a brief induction in UTA-L cells is sufficient to observe its targeting to mid, late, and at least certain early replication foci visualized by a pulse-labeling with bromodeoxyuridine (BrdU). In addition, when UTA-L cells are simultaneously induced for BCL6 expression and exposed to BrdU for a few hours just after the release from a block in mitosis, a nuclear diffuse BCL6 staining indicates cells in G(1), while cells in S show a more punctate nuclear BCL6 distribution associated with replication foci. Finally, ultrastructural analyses in UTA-L cells exposed to BrdU for various times reveal that replication progresses just around, but not within, BCL6 subdomains. Thus, nascent DNA is localized near, but not colocalized with, BCL6 subdomains, suggesting that they play an architectural role influencing positioning and/or assembly of replication foci. Together with its previously function as transcription repressor recruiting a histone deacetylase complex, BCL6 may therefore contribute to link nuclear organization, replication, and chromatin-mediated regulation.

Isolation and characterization of an equine foamy virus.

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway.

An anchorage nuclear structure for telomeric DNA repeats in HeLa cells.

Using either a biotinylated peptide nucleic acid (PNA) oligomer or a digoxigenin-labeled double-stranded DNA probe, we determined the distribution of the telomeric DNA repeats in HeLa cells by in-situ hybridization at the ultrastructural level. The telomeric DNA was found at the periphery of previously unrecognized roundish nuclear structures, distributed throughout the nucleoplasm. The levels of association of the telomeric DNA with these structures was investigated by exposure of cells to a detergent-containing hypotonic solution which only preserves tightly linked components. The telomeric DNA repeats stayed associated with their anchorage structures following spreading apart of nucleoproteins. Because changes in cellular DNA topology are associated with the intranuclear development of herpes simplex virus type 1 (HSV-1) and adenovirus type 5 (Ad5) in HeLa cells, we examined the distribution of telomeric DNA when cellular DNA is pushed toward the nuclear border. The circular telomeric complexes were morphologically unmodified; however, as a result of the partition of cellular and viral DNA in two concentric compartments, they migrated towards the nuclear border, along with the compressed cellular chromatin. Taken together, our results exemplify the unique organization of the telomeric DNA, which is coiled around a central core of a diameter of 120 nm and can therefore be clearly distinguished from the bulk of the cellular chromatin.

Deletion of the fiber gene induces the storage of hexon and penton base proteins in PML/Sp100-containing inclusions during adenovirus infection.

The present study has documented changes in the in situ distribution of viral DNA and capsid proteins in 293 cells infected with fiber gene-deleted adenoviruses. It shows that infection results in the intense production of progeny viruses which appear morphologically intact although they are devoid of fiber-coding sequence in their genome and hence of fiber protein in their capsid. The data confirm, therefore, that fiber protein is not essential for the assembly of progeny viruses. The main contribution of our observations concerns specific intranuclear structures induced by infection with either wild-type or fiber gene-deleted viruses. These clear amorphous inclusions contain two cellular proteins, PML and Sp100, which in non-infected cells co-localize to a special type of nuclear bodies. PML and Sp100 nuclear bodies appear to directly modulate or to be altered in a wide variety of situations including viral infections, cell death and transformation. In cells infected with fiber gene-deleted viruses, the clear amorphous inclusions now accumulate non-used hexon and penton base proteins, whereas the absence of fiber protein prevents the assembly of capsid proteins in crystallin arrays. Taken together, the data suggest that the clear amorphous inclusions may correspond to storage sites of structural and regulatory proteins. Consequently, these virus-induced structures may promote the productive cycle of adenoviruses by regulating the amount of over-produced viral proteins and the shutoff of the host cell metabolism.

Release of viruses and viral DNA from nucleus to cytoplasm of HeLa cells at late stages of productive adenovirus infection as revealed by electron microscope in situ hybridization.

Considerable progress has been made over the past 10 years towards a full understanding of the functional significance of the structural changes resulting from the production of adenoviruses in permissive cells. Similarly, the host-virus interactions which are involved in viral replication and gene expression as well as in RNA nuclear export have been investigated. Post-embedding nonisotopic in situ hybridization has been proven to be a powerful tool for the study of nucleic acids in infected cells provided that controlled elimination of artifacts by appropriate treatments was undertaken. Adenovirus infected cells present two biological characteristics which could lead to false positive or negative results. First, they contain large amounts of single-stranded portions of viral DNA which are revealed with viral RNA molecules. Second, DNA-binding proteins are present which hide some nucleic acid sequences. By using a DNA probe and appropriate variations in the experimental protocol, it is possible to reveal specifically different kinds of targets, simultaneously single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), or only ssDNA, or only dsDNA, or only RNA. By using two probes labeled with different haptens, biotine and digoxigenin, it is possible to detect concomitantly two nucleic acid targets and, therefore, to study their relationships. In order to gain insight into the changes in the nucleus before cell lysis and to improve our knowledge on the series of steps leading to the release of adenoviruses from the nucleus, examination of cells at 41 h post-infection and identification of structures containing adenoviral nucleic acids were undertaken. In addition to the ultrastructural changes and precise distribution of cellular DNA and viral nucleic acid molecules already described in cells up to 24 h post-infection (for a review, see Puvion E, Puvion-Dutilleul F (1996) Exp Cell Res 229, 217-225), new results were obtained. Routine observation revealed the presence of: i) viruses in the cytoplasm, some being located next to nuclear pores; ii) abnormally large portions of the nuclear envelope devoid of underlying condensed chromatin; iii) proliferation of either the inner nuclear membrane only or both membranes of the nuclear envelope; and iv) electron-opaque grains in the nuclear compartment involved in viral genome transcription, and also in the clusters of interchromatin granules known to contain mature viral messenger RNA (Bridge E et al (1996) J Cell Biol 135, 303-314). In situ hybridization revealed the presence of: i) dsDNA in the cytoplasmic viruses indicating that they were mature viruses; ii) free viral dsDNA and ssDNA molecules in the cytoplasm whereas host DNA remained confined at the nuclear border; and iii) viral RNA in the newly-described electron-opaque grains we call, therefore, viral-RNA containing grains. Immunodetection of bromodeoxyuridine (BrdU) incorporated into DNA in pulse and pulse-chase experiments allowed us to ascertain that cells at 41 h post-infection were truly living cells and that at least part of the newly-synthesized viral DNA migrated from the nucleus to the cytoplasm. Taken together, the data suggest that modifications of the nuclear matrix, cytoskeleton, and nucleo-cytoplasmic interactions might occur near the termination of adenovirus infection inducing the progressive release of viruses, vial dsDNA and ssDNA molecules in the cytoplasm. In addition, the observation of a new structural support for the intranuclear viral RNA in the clusters of interchromatin granules emphasizes the role of these cellular structures in the intranuclear trafficking of messenger RNA leading to the regulation of its nuclear export.

Pilot ultrastructural evaluation of human preauricular skin before and after high-energy pulsed carbon dioxide laser treatment.

BACKGROUND: Carbon dioxide laser resurfacing has recently come into favor for the treatment of photodamaged skin. While the clinical and histologic effects of high-energy short-pulse carbon dioxide lasers on human skin have been investigated, the ultrastructural effects of these lasers have not been documented. Our objective was to study the ultrastructural effects of a high-energy pulsed carbon dioxide laser on photodamaged human skin. OBSERVATIONS: Before laser surgery, the ultrastructural changes characteristic of photodamaged skin were evident. Immediately after treatment, there was extensive coagulation necrosis of the epidermis and papillary dermis. Thirty days after treatment, there was no evidence of intercellular or intracellular edema, and ordered differentiation of the epidermal keratinocytes, with a loss of keratinocyte dysplasia, was seen. Increased numbers of desmosomes and tonofibrils were noted. New deposition of collagen was present in the papillary dermis. The ultrastructural findings seen at 90 days after treatment were similar to those seen at 30 days, apart from increased organization of collagen fibers in the papillary dermis. CONCLUSIONS: Treatment with the high-energy pulsed carbon dioxide laser appears to reverse the epidermal and dermal changes of photoaging on an ultrastructural level. These changes appear morphologically to be consistent with previously described clinical and histologic changes following laser resurfacing.

Ultrastructural localization of interferon-inducible double-stranded RNA-activated enzymes in human cells.

The protein kinase PKR and the 2',5'-oligoadenylate (2-5A) synthetase are two interferon-induced and double-stranded RNA-activated enzymes which are implicated in the mechanism of action of interferon. Their distribution was undertaken here at the ultrastructural level by the immunogold procedure, following the use of specific monoclonal antibodies directed against PKR and 69- and 100-kDa forms of the 2-5A synthetase. These enzymes were detected as a pool of nonaggregated proteins scattered throughout the cell and as aggregates often associated with electron-dense doughnut-like structures showing a similar aspect whatever their subcellular localization: the cytoplasm, the nuclear envelope, and the nucleus. In general, the 2-5A synthetases were present in much more lower amounts than the PKR, probably due to the difficulty of detecting traces of proteins by electron microscopy. To circumvent this, we used a human lymphoblastoid cell line overexpressing the 69-kDa form of the 2-5A synthetase. In such cells, the synthetase was then clearly observed in both the cytoplasm and the nucleus; isolated or small clusters of gold particles were numerous in the cell mainly over the RNP fibrils of the interchromatin space, nucleolus, and ribosomes. Interestingly, gold particles were also found to be associated with the membranes of nuclear envelope and rough endoplasmic reticulum probably due to the myristilated motif of this form of 2-5A synthetase. Finally, intensely labeled electron-opaque dots sometimes associated with the nuclear pore complexes were present in the nucleus and in the cytoplasm of cells which might suggest their transport from the nucleus to the cytoplasm or reciprocally through the nuclear pore complexes. These observations indicate the wider distribution of the dsRNA-activated enzymes in the cell, thus pointing out their potential implication in as yet undetermined physiological function(s) necessary for various cellular metabolic reactions.

Early stages of pre-rRNA formation within the nucleolar ultrastructure of mouse cells studied by in situ hybridization with a 5'ETS leader probe.

The first cleavage in the processing of the rRNA primary transcript in mammals occurs within the 5'-terminal region of the 5' external transcribed spacer (5'ETS), which makes the upstream portion of this spacer a selective marker of nascent transcripts. Moreover, short treatments with low doses of actinomycin D (AMD), which selectively suppress pre-rRNA synthesis and allow processing of preformed pre-rRNAs, result in the production of prematurely terminated transcripts essentially spanning the 5'ETS leader region. To gain further insight into the intranucleolar localization of early stages of preribosome formation we analyzed the distribution of this specific pre-rRNA segment by in situ hybridization at the ultrastructural level in AMD-treated or in control 3T3 mouse cells. In control cells, 5'ETS leader rRNA was detected at the border of the fibrillar centers and over the dense fibrillar component, in agreement with previous data suggesting that rRNA gene transcription takes place at the border of the fibrillar centers before a rapid transfer of the nascent trancript to the dense fibrillar component. Observation of cells subjected to a short treatment with low doses of AMD fully supports this conclusion, with the prematurely terminated 5'ETS leader-containing transcripts detected at the border of enlarged fibrillar centers. With prolonged periods of AMD treatment even the partial transcription of rRNA genes is blocked and fibrillar centers of typically segregated nucleoli show no positive signals with the 5'ETS leader probe. We also analyzed in parallel the intranucleolar distribution of U3 small nucleolar RNA, which is involved in 5'ETS processing, by hybridization with biotinylated antisense oligonucleotides. Distribution of U3 roughly paralleled that of 5'ETS leader rRNA in untreated cells. However, U3 RNA persisted in the dense fibrillar component of segregated nucleoli whatever the conditions of drug treatment, i.e., even after a thorough chase of the rRNA precursors from this nucleolar compartment.

The ultrastructural localization of 60-kDa Ro protein and human cytoplasmic RNAs: association with novel electron-dense bodies.

The 60-kDa Ro ribonucleoprotein is an important target of humoral autoimmune responses. However, the ultrastructural locations of the 60-kDa Ro protein and its associated small cytoplasmic RNAs (Y RNAs) have not been previously determined, and the functions of the Ro protein and RNAs are not known. In this study, the cellular locations of the 60-kDa Ro protein and the Ro Y1 and Y4 RNAs are determined by immunoelectron microscopy and in situ hybridization electron microscopy, respectively. Both Ro protein and Y RNAs are concentrated in discrete areas of the nucleoplasm, nucleolus, and cytoplasm of cultured cells and human skin sections. The 60-kDa Ro protein and Y RNAs are also present diffusely in the cytoplasm, where they occur in ribosome-rich regions, and in the nucleus. The presence of Ro ribonucleoprotein components in nucleoli and in ribosome-rich cytoplasmic areas suggests a potential for the involvement of Y RNAs and/or 60-kDa Ro protein in ribosome synthesis, assembly, or transport. Double labeling experiments show that Ro protein and Y RNAs colocalize in the nucleoplasm, nucleolus, and cytoplasm. In addition, aggregates of Y RNA occur unassociated with 60-kDa Ro protein, and aggregates of 60-kDa Ro protein occur unassociated with Y RNA. Aggregates of both Ro protein and Y RNAs label previously unreported nuclear and cytoplasmic electron-dense bodies. We propose that these distinctive Ro-associated electron-dense bodies may represent structure(s) important for cellular transport and/or Ro function.

Nuclear targeting of incoming human foamy virus Gag proteins involves a centriolar step.

The pathways used in the transport of retroviral genomes to the nucleus are poorly identified. Analyzing the intracellular localization of incoming foamy viruses, we have found that the Gag antigens and the viral genome accumulate in a distinct perinuclear domain identified as the centrosome. Colchicine treatment completely abolished pericentriolar targeting of human foamy virus (HFV) proteins, suggesting a role for microtubules in the transport of the incoming viral proteins to the centrioles. Finally, we demonstrate that, similarly to human immunodeficiency virus DNA, HFV DNA can enter the nucleus of G1/S-phase-arrested cells, although no viral gene expression can be observed. Recent observations have demonstrated that foamy viruses have several features not shared by other retroviruses. The intracellular route of the incoming Gag antigens may constitute a new specificity of this class of viruses.

Identification of transcription factories in nuclei of HeLa cells transiently expressing the Us11 gene of herpes simplex virus type 1.

Nuclear distribution and migration of herpes simplex virus type 1 Us11 transcripts were studied in transient expression at the ultrastructural level and compared to that of RNA polymerase II protein. Transcription was monitored by autoradiography following a short pulse with tritiated uridine. Us11 transcripts accumulated mainly over the foci of intermingled RNP fibrils as demonstrated by the presence of silver grains localizing incorporated radioactive uridine superimposed to these structures in which the presence of Us11 RNA and poly(A) tails was previously demonstrated. Silver grains were also scattered over the remaining nucleoplasm but not in the clusters of interchromatin granules, and over the dense fibrillar component of the nucleolus as in control, nontransfected HeLa cells. Pulse-chase experiments revealed the transient presence of migrating RNA in the clusters of interchromatin granules. RNA polymerase II was revealed by immunogold labeling following the use of two monoclonal antibodies: mAb H5, which recognizes the hyperphosphorylated form of the carboxy-terminal domain (CTD) of the molecule, and mAb 7C2, which recognizes both its hyperphosphorylated and unphosphorylated forms. The two mAbs bind to the newly formed Us11 transcription factories and the clusters of interchromatin granules of transfected cells. In control cells, however, clusters of interchromatin granules were labeled with mAb H5 but not with mAB 7C2. Taken together, our data demonstrate the involvement of the clusters of interchromatin granules in the intranuclear migration of Us11 RNA in transient expression. They also suggest the occurrence of changes in the accessibility of the RNA polymerase II CTD upon expression of the Us11 gene after transfection by exposing some epitopes, otherwise masked in nontransfected cells.

Ultrastructure of the nucleus in relation to transcription and splicing: roles of perichromatin fibrils and interchromatin granules.

In the present article we summarize our results concerning the in situ ultrastructural organization of the nuclear steps of mRNA production using several cellular models including normal noninfected cells, herpes simplex virus type 1 and adenovirus type 5 infected cells, and transiently transfected cells. We confirm that perichromatin fibrils are the in situ morphological expression of nascent transcripts and the main support of splicing. Special emphasis is devoted to the clusters of interchromatin granules which seem to be involved in several central functions including accumulations of snRNP components as well as sorting and/or regulation of export of different RNA species.

Localization of the newly initiated and processed ribosomal primary transcripts during the mitotic cycle in Physarum polycephalum.

We analyzed the fate of the rRNA released as a result of the prophasic disintegration of the nucleolus, during the "closed" mitosis of the naturally synchronous plasmodium of Physarum polycephalum. Using a probe complementary to the mature 19S and 26S rRNA, we previously showed that the nucleolus-derived rRNAs are stable in mitosis, mainly associated with numerous fibrillar nucleolar remnants (Pierron and Puvion-Dutilleul, 1993, Exp. Cell Res. 208, 509-517). However, a significant fraction of these mitotic rRNA precursors were also found in more diffuse nuclear, granular zones. In this paper, we trace pre-rRNA molecules in the early stages of their maturation by using probes derived from the 5' external transcribed spacer (5'-ETS), upstream and downstream of the processing site located 1.7 kb from the transcription initiation site. In agreement with a previous S1-mapping study (Blum et al., 1986, Nucleic Acids Res., 10, 4121-4133), we observed that the rRNA transcripts complementary to the 5'-ETS portion downstream of the processing site are stable in mitosis, and we demonstrate that they are specifically associated with the border of the fibrillar nucleolar remnants. On the other hand, the pre-rRNA chains containing the upstream portion of the 5'-ETS become undetectable in mitosis, marking the cessation of rDNA transcription. Since the reappearance of these nascent pre-rRNA molecules signals the resumption of the transcriptional activity, we determined that a burst of rRNA synthesis is taking place within the prenucleolar bodies, immediately after the separation of the daughter nuclei. Taken together, our results illustrate the persistence of the nucleolar components of Physarum, within nucleolar remnants that, although transcriptionally inactive during mitosis, function as independent entities very early in interphase.

Distribution of ribosomal genes in nucleoli of herpes simplex virus type 1 infected cells.

Herpes simplex virus type 1 (HSV-1) infection of HeLa cells induces profound changes in the structure of the nucleoli. They become markedly elongated, and their fibrillar centers become greatly diminished in number, but larger than in non-infected HeLa cells, and only partially surrounded by the dense fibrillar component. The effect of prolonged HSV-1 infection on the distribution of the rRNA genes was studied by means of postembedding electron microscope in situ hybridization using a biotinylated ribosomal DNA (rDNA) probe, which spans about half of the rRNA gene, and subsequent immunogold labeling of the resulting hybrids. Gold particles accumulated over two structures: a large solitary, finely fibrillar, moderately electron opaque area which was detectable only in a few sections of nucleoli and corresponded to the virus-modified fibrillar center, and over limited areas of the nucleolus-associated chromatin. In non-infected HeLa cells, foci of clustered rRNA genes were observed in the more frequently detected fibrillar centers and in association with condensed chromatin. It would be expected that foci of extended rDNA molecules might contain active or potentially active genes, whereas foci of highly compacted rDNA molecules might contain inactive genes. The ribosomal RNA molecules which were detected with the same probe over the dense fibrillar component and the granular component of the nucleoli of both infected and non-infected cells were not found within the rDNA-containing foci. The data strongly suggest that the changes in the size and number of fibrillar centers induced by the intranuclear development of HSV-1 might be directly linked to the well-known decrease of the nucleolar activity.

LYSP100-associated nuclear domains (LANDs): description of a new class of subnuclear structures and their relationship to PML nuclear bodies.

The PML gene is fused to the retinoic acid receptor alpha (RAR alpha) gene in t(15;17) acute promyelocytic leukemia (APL), creating a PML-RAR alpha fusion oncoprotein. The PML gene product has been localized to subnuclear dot-like structures variously termed PODs, ND10s, Kr bodies, or PML nuclear bodies (PML NBs). The present study describes the cloning of a lymphoid-restricted gene, LYSP100, that is homologous to another protein that localizes to PML NBs, SP100. In addition to SP100 homology regions, one LYSP100 cDNA isoform contains a bromodomain and a PHD/TTC domain, which are present in a variety of transcriptional regulatory proteins. By immunofluorescence, LYSP100 was localized to nuclear dots that were surprisingly largely nonoverlapping with PML NBs. However, a minority of LYSP100 nuclear dots exactly colocalized with PML and SP100. We term the LYSP100 structures "LANDs," for LYSP100-associated nuclear domains. Although LYSP100 is expressed only in lymphoid cells, LANDs could be visualized in HeLa cells by transfection of a LYSP100 cDNA. Immunoelectron microscopy revealed LANDs to be globular, electron-dense structures morphologically distinct from the annular structures characteristic of PML NBs. LANDs were most often found in the nucleoplasm, but were also found at the nuclear membrane and in the cytoplasm, suggesting that these structures may traffic between the cytoplasm and the nucleus. By double-immunogold labeling of PML and LYSP100, some LANDs were shown to contain both PML and LYSP100. Thus, PML is localized to a second subnuclear domain that is morphologically and biochemically distinct from PML NBs.

Nucleolin is a sequence-specific RNA-binding protein: characterization of targets on pre-ribosomal RNA.

Nucleolin is an abundant nucleolar protein, which plays an essential, but largely unknown role in ribosome biogenesis. Nucleolin contains four consensus RNA-binding domains (CS-RBD), the presence of which suggests that the molecular function of this protein is likely reflected by its RNA-binding properties. Indeed, by immunocytological analysis performed on ribosomal transcription units, we have found several nucleolin molecules associated with nascent pre-rRNA. In mouse, two high-affinity binding sites with an apparent dissociation constant (Kd) of 50 to 100 nM have been mapped in the 5' ETS upstream from the early pre-rRNA processing site. Interestingly, nucleolin of mouse origin has recognized analogous sequences in the 5' ETS of human pre-rRNA. In parallel, selection-amplification (SELEX) experiments have identified an 18-nucleotide long RNA sequence that binds nucleolin with high affinity (Kd 5 to 20 nM) and shares a common UCCCGA motif with the characterized pre-rRNA binding sites. By mutagenesis and a structural analysis, we have characterized the nucleolin RNA binding site and found that it is constituted by a minimal 18-nucleotide long stem-loop structure. The sequence UCCCGA that is found within the hairpin loop is necessary for the specific interaction. Mutation of any of the C or G residues within this motif abolishes nucleolin interaction. Furthermore, point mutation in the stem that completely disrupt the hairpin structure also prevents nucleolin binding. By determining the minimal 5' and 3' ends of the RNA that is bound to the protein we concluded that nucleolin binding site is constituted by a short four to five-base-pair stem and an eight-nucleotide loop. This structural motif is very similar to hairpins recognized by two other CS-RBD-containing proteins (U1 snRNP A and U2 snRNP B"). Possible functional implications of our findings are discussed.

Non-isotopic electron microscope in situ hybridization for studying the functional sub-compartmentalization of the cell nucleus.

Post-embedding electron microscope in situ hybridization using gold particles as label permits the clear identification of the cellular structures which contain the nucleic acid molecules under study. It has yielded information on the distribution of defined nucleic acid sequences of different origins-cellular or viral, DNA or RNA, single- or double-stranded molecules-which has revolutionized the study of the nucleus. Application of this powerful technique in combination with other refined techniques to studies on transcription and replication of cellular and viral genes has augmented our knowledge of the functional organization of the cell nucleus. One can now ask mechanistically meaningful questions concerning the successive steps of gene replication and expression not only under normal conditions of cell growth, but also when the cellular metabolism is altered by a drug treatment or a viral infection. This chapter aims (a) to present the established methods of post-embedding electron microscope in situ hybridization for localizing, precisely and specifically, a nucleic acid target in its normal environment and (b) to present some contributions of this technique to investigations of the functional compartmentalization of the cell nucleus and to elucidate the cell-virus relationships in infected cells.

Nucleoplasmic and nucleolar distribution of the adenovirus IVa2 gene product.

Sequence elements (DE) located downstream of the adenovirus major late promoter start site have previously been shown to be essential for the activation of this promoter after the onset of viral DNA replication. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner and contribute to this activation. DEF-B corresponds to a dimer of the adenovirus IVa2 gene product (pIVa2, 449 residues), while DEF-A is a heteromeric protein also comprising pIVa2. As revealed by specific immunofluorescence staining of infected cells, pIVa2 is targeted to the nucleus, where it distributes to both nucleoplasmic and nucleolar structures. We have identified the pIVa2 nuclear localization signal (NLS) as a basic peptide element at the C terminus of the protein (residues 432 to 449). An element essential for nucleolar localization (NuLS) has been mapped in the N-terminal part of pIVa2 (between residues 50 and 136). While NuLS activity is dependent upon an intact NLS, we show that both NLS and NuLS functions are independent of specific DNA-binding activity. As visualized by immunoelectron microscopy, pIVa2 is detected in the nucleoplasm at the level of the fibrillogranular network which is active in viral transcription. More surprisingly, pIVa2 accumulates within electron-dense amorphous inclusions found both in the nucleoplasm and in the nucleolus. Altogether, these results suggest that, besides controlling major late promoter transcription, pIVa2 serves additional, as yet unknown functions.