Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro

RNA synthetic activity of human 2-16-cell embryos developing in vitro was studied by [3H]uridine light-microscope autoradiography. Parallelly cut thin sections were examined in the electron microscope. The first extranucleolar RNA synthesis was detected in 4-cell embryos, but nucleoli were never labelled until the 3rd cleavage (6-8-cell embryos). In 6-cell embryos the nucleolar labelling was mostly confined to a narrow peripheral zone. In later cleavage stages most of the blastomeres showed intensive labelling of nucleoli and extranucleolar chromatin. However, rather low levels of extranucleolar RNA synthesis and the absence of nucleolar activity were often seen even in blastomeres of fully compacted morulae. The activation of nucleolar RNA synthesis entailed a noticeable increase in the number of ribosomes (estimated by electron microscope morphometry) that followed a marked drop during the period between the 2-cell and 8-cell stages. The results indicate that the concentration of ribosomes in the preovulatory oocyte is a major factor of its developmental potential.     

Detection of fibrillarin in nucleolar remnants and the nucleolar matrix

In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.     

Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast

NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo.     

Composition and structure of nucleolar skeleton (nucleolar matrix)——Actin and fibrillarin are two main protein components of nucleolar skeleton

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6-7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fib-rillarin are two major protein components of nucleolar skeleton of HeLa cells.     

Modulation of collagen metabolism by the nucleolar protein fibrillarin.

Metabolic functions of fibroblasts are tightly regulated by the extracellular environment. When cultivated in tridimensional collagen lattices, fibroblasts exhibit a lowered activity of protein synthesis, especially concerning extracellular matrix proteins. We have previously shown that extracellular collagen impaired the processing of ribosomal RNA (rRNA) in nucleoli by generating changes in the expression of nucleolar proteins and a premature degradation of neosynthesized rRNA. In this study, we have investigated whether inhibiting the synthesis of fibrillarin, a major nucleolar protein with decreased expression in collagen lattices, could mimic the effects of extracellular matrix. Monolayer-cultured fibroblasts were transfected with anti-fibrillarin antisense oligodeoxynucleotides, which significantly decreased fibrillarin content. Downregulation of fibrillarin expression inhibited procollagen secretion into the extracellular medium, without altering total collagen production. No changes of pro1(I)collagen mRNA expression or proline hydroxylation were found. A concomitant intracellular retention of collagen and its chaperone protein HSP47 was found, but no effect on the production of other extracellular matrix macromolecules or remodelling enzymes was observed. These data show that collagen processing depends on unknown mechanisms, involving proteins primarily located in the nucleolar compartment with other demonstrated functions, and suggest specific links between nucleolar machinery and extracellular matrix.     

Composition and structure of nucleolar skeleton (nucleolar matrix)-Actin and fibrillarin are two main protein components of nucleolar skeleton

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6–7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fibrillarin are two major protein components of nucleolar skeleton of HeLa cells.     

Analysis of nucleolar protein fibrillarin mobility and functional state in living HeLa cells

Fibrillarin is an evolutionarily-conserved and obligatory protein component of eukaryotic cell nucleoli involved in pre-rRNA processing and methylation. In vertebrates the fibrillarin molecule contains two cysteine residues (Cys99 and Cys268) whose sulfhydryl groups are able to establish intramolecular -S-S- bridges. However, the functional state of fibrillarin with reduced or oxidized thiol groups is still practically unstudied. Besides, there are no data in the literature concerning existence of the -S-S- fibrillarin form in human cells. To answer these questions, we used plasmids encoding native human fibrillarin and its mutant form devoid of cysteine residues (fibrillarinC99/268S) fused with EGFP for temporary transfection of HeLa cells. The mobile fraction localizing the enzymatically active protein molecules and the fluorescence half-recovery time characterizing the rate of enzymatic reactions were determined by the FRAP technique using a confocal laser scanning microscope. Measurements were carried out at 37 and 27°C. The results show that the fibrillarin pool in HeLa cells includes two protein forms, with reduced SH groups and with oxidized SH groups forming intramolecular -S-S- bridges between Cys99 and Cys268. However, the absence of Cys99 and Cys268 has no effect on intracellular localization of fibrillarin and its main dynamic parameters. The human fibrillarin form without disulfide bridges is included into the mobile protein fraction and is consistent with its functionally active state.     

A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro

Fibrillarin, one of the major proteins of the nucleolus, has methyltransferase activity directing 2′-O-ribose methylation of rRNA and snRNAs and is required for rRNA processing. The ability of the plant umbravirus, groundnut rosette virus, to move long distances through the phloem, the specialized plant vascular system, has been shown to strictly depend on the interaction of one of its proteins, the ORF3 protein (protein encoded by open reading frame 3), with fibrillarin. This interaction is essential for several stages in the groundnut rosette virus life cycle such as nucleolar import of the ORF3 protein via Cajal bodies, relocalization of some fibrillarin from the nucleolus to cytoplasm, and assembly of cytoplasmic umbraviral ribonucleoprotein particles that are themselves required for the long-distance spread of the virus and systemic infection. Here, using atomic force microscopy, we determine the architecture of these complexes as single-layered ringlike structures with a diameter of 18-22 nm and a height of 2.0 ± 0.4 nm, which consist of several (n = 6-8) distinct protein granules. We also estimate the molar ratio of fibrillarin to ORF3 protein in the complexes as approximately 1:1. Based on these data, we propose a model of the structural organization of fibrillarin-ORF3 protein complexes and discuss potential mechanistic and functional implications that may also apply to other viruses.     

Small nucleolar RNAs and nucleolar proteins inXenopus anucleolate embryos

We investigated the presence and localization, in the cells of anucleolate mutant embryos of Xenopus laevis, of three representative small nucleolar RNAs (snoRNAs), U3, U15 and U17, and of two nucleolar proteins, nucleolin and fibrillarin. The levels of the three snoRNAs in the anucleolate mutant are the same as in normal embryos, in contrast to 5S RNA and ribosomal proteins. In situ hybridization showed that, in the absence of fully organized nucleoli, the three RNAs are diffusely distributed in the nucleus and partly associated with a number of small structures. Nucleolin and fibrillarin are also present in the anucleolate embryos as in normal embryos, although there is less nucleolin mRNA in the former. The two nucleolar proteins were localized by immunofluorescence microscopy. Fibrillarin, similar to its associated U3 and U15 snoRNAs, is diffusely distributed in the anucleolate nucleus and is partly associated with small structures, probably prenucleolar bodies and pseudonucleoli. Nucleolin also appears diffusely distributed in the nucleus with some spots of higher concentration, but with a different pattern with respect to fibrillarin. Received: 26 September 1996; in revised form: 14 February 1997 / Accepted: 24 February 1997     

Onset of paternal and maternal Gpi-1 expression in preimplantation mouse embryos

The initial activation of the glucose phosphate isomerase gene, Gpi-1, was studied in mouse embryos produced by transplanting pronuclei between two strains of mice differing in alleles for this enzyme. Protein isozymes encoded by the embryonic cell nuclei were first detected on Day 4 of embryogenesis, and the maternal and paternal genes are seen to be activated simultaneously. Comparison of isozymes produced by these nuclear-transfer embryos and by F1 embryos from these two strains suggests the absence of oocyte mRNA for GPI-1 at the time when these genes are first activated. Thus, the GPI-1 present is derived from newly transcribed mRNA contributed by both maternal and paternal genes. The relative proportion of maternal cytoplasmic GPI-1 enzyme declines from Day 3 to Day 6, such that on Day 6, almost no oocyte GPI-1 is detected.     

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.