Two Splice Variants of Nopp140 in Drosophila Melanogaster.

The activities of non-ribosomal nucleolar proteins are now understood to be important for the normal functions of both nucleoli and C&barbelow;ajal B&barbelow;odies (CBs). Although these proteins have been studied extensively in other eukaryotes, knowledge of non-ribosomal nucleolar proteins in Drosophila melanogaster lags far behind. The n&barbelow;ucleo&barbelow;lar p&barbelow;hosphop&barbelow;rotein of 140 kDa (Nopp140) may function to shuttle box C/D and box H/ACA s&barbelow;mall n&barbelow;ucleo&barbelow;lar (sno)RNAs from the nucleus to the nucleolus, where they function in the 2 \u27-O-methylation and pseudouridylation of rRNA, respectively. Nopp140 homologues have been described in rat, human, Xenopus laevis , and yeast. This dissertation describes the cloning of cDNAs that encode two splice variants of Nopp140 in D. melanogaster. In addition, this dissertation addresses the localization patterns of the D. melanogaster Nopp140 splice variants in various cell types with respect to endogenous nucleolar proteins and CBs. The D. melanogaster Nopp140 gene maps within 79A5 of chromosome 3. Alternative mRNA splicing yields two variants. DmNopp140 (654 residues) is the true D. melanogaster homologue of vertebrate Nopp140 in that its carboxy terminus is 58% identical to the carboxy terminus of rat Nopp140. DmNopp140-RGG (688 residues) is identical to DmNopp140 throughout its first 551 residues, but its carboxy terminus contains an extensive ar&barbelow;rginine-g&barbelow;lycine-g&barbelow;lycine (RGG) domain that is found in many RNA-binding proteins such as vertebrate nucleolin. Both D. melanogaster Nopp140 variants localize to the dense fibrillar component (DFC) of D. melanogaster Schneider II cells and X. laevis oocytes. In HeLa cells, DmNopp140-RGG localizes to intact nucleoli, while DmNopp140 segregates nucleoli into phase-light and phase-dark regions. The phase light regions contain DmNopp140 and endogenous fibrillarin, while the phase-dark regions contain endogenous nucleolin. Both D. melanogaster variants co-localize to nucleoli when co-expressed in HeLa cells. Both proteins also co-localize with exogenously expressed X. laevis coilin to enlarged C&barbelow;ajal b&barbelow;odies (CBs) within HeLa cell nucleoli, but only DmNopp140 localizes to CBs in Schneider II cells. Both variants fail to localize to CBs in X. laevis oocyte nuclei. A carboxy terminal truncation, DmNopp140-DeltaRGG, fails to localize to nucleoli in HeLa cells, but like DmNopp140, it localizes with exogenously expressed coilin in HeLa cell CBs

Genomic organization of nucleolin gene in carp fish: Evidence for several genes

http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000200017&lng=es&nrm=isoThe protein nucleolin, functionally involved in the main steps of ribosome biogenesis, is codified by a single copy gene in mammals. Here we report that at least three different genes codify for this protein in carp fish (Cyprinus carpio). This is the first description of the genomic organization of nucleolin in a teleost. The carp nucleolin gene includes 8.8 kb and contains 16 exons. Promoter cis regulatory elements are similar to constitutive genes, i.e., a putative TATA box, three G/C boxes, and three pyrimidine-rich boxes. As in other species, carp nucleolin gene introns host three snoRNA codifying sequences: U23 from the H/ACA family and two C/D box snoRNAs, U20 and U82. Both U20 and U82 span a complementary sequence with carp 18S rRNA. Additionally, we identified two cDNAs coding for nucleolin, confirming the existence of several nucleolin genes in carp. Amino acidderived sequence from carp cDNAs differ from mammal protein because they span additional acidic domains at the amino end, whose functional significance remains unclear. We performed amino acid sequence comparison and phylogenetic analyses showing that the three isoforms of carp nucleolin, which we describe herein, cluster in two groups. cNUC1 probably diverges from cNUC2 and cNUC3 as result of ancestral fish-specific genome duplication, indeed C. carpio is a tetraploid fish

Nucleolin: a multifunctional major nucleolar phosphoprotein

Nucleolin is a major protein of exponentially growing eukaryotic cells where it is present in abundance at the heart of the nucleolus. It is highly conserved during evolution. Nucleolin contains a specific bipartite nuclear localization signal sequence and possesses a number of unusual structural features. It has unique tripartite structure and each domain performs a specific function by interacting with DNA or RNA or proteins. Nucleolin exhibits intrinsic self-cleaving, DNA helicase, RNA helicase and DNA-dependent ATPase activities. Nucleolin also acts as a sequence-specific RNA binding protein, an autoantigen, and as the component of a B cell specific transcription factor. Its phosphorylation by cdc2, CK2, and PKC-zeta modulate some of its activities. This multifunctional protein has been implicated to be involved directly or indirectly in many metabolic processes such as ribosome biogenesis (which includes rDNA transcription, pre-rRNA synthesis, rRNA processing, ribosomal assembly and maturation), cytokinesis, nucleogenesis, cell proliferation and growth, cytoplasmic-nucleolar transport of ribosomal components, transcriptional repression, replication, signal transduction, inducing chromatin decondensation and many more (see text). In plants it is developmentally, cell-cycle, and light regulated. The regulation of all these functions of a single protein seems to be a challenging puzzle

Identification of a novel nucleolin related protein (NRP) gene expressed during rat spermatogenesis

<p>Abstract</p> <p>Background</p> <p>Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells.</p> <p>Results</p> <p>By RT PCR and western blot analysis we found that nucleolin is strongly down regulated in meiotic spermatocytes and haploid germ cells. We have identified a new nucleolin related protein (NRP) gene in the rat genome, which is over expressed in the testis and is up regulated several fold in meiotic spermatocytes and haploid germ cells. The NRP protein lacks the acidic stretches in its N terminal domain, and it is encoded in rat chromosome 15 having a different genomic organization as compared to nucleolin gene present on chromosome 9. We have also found NRP genes encoded in genomes of other mammalian species. We performed run-on transcription assay where we have observed that rDNA is transcribed at much lower level in meiotic spermatocytes and haploid spermatids as compared to diploid cells. By siRNA knock down experiments we could also demonstrate that NRP can support rDNA transcription in the absence of nucleolin.</p> <p>Conclusion</p> <p>We have identified a new nucleolin variant over expressed in germ cells in rat and analyzed its domain structure. We attribute that the transcriptional activity of rDNA genes in the late spermatogenesis is due to the presence of this variant NRP. The expression of this variant in the germ cells in the absence of nucleolin, could have additional functions in the mammalian spermatogenesis which needs to be investigated further.</p

The Scd6/Lsm14 protein xRAPB has properties different from RAP55 in selecting mRNA for early translation or intracellular distribution in Xenopus oocytes

© 2015 Elsevier B.V. Oocytes accumulate mRNAs in the form of maternal ribonucleoprotein (RNP) particles, the protein components of which determine the location and stability of individual mRNAs prior to translation. Scd6/Lsm14 proteins, typified by RAP55, function in a wide range of eukaryotes in repressing translation and relocating mRNPs to processing bodies and stress granules. In Xenopus laevis, the RAP55 orthologue xRAPA fulfils these functions. Here we describe the properties of a variant of xRAPA, xRAPB, which is a member of the Lsm14B group. xRAPB differs from xRAPA in various respects: it is expressed at high concentration earlier in oogenesis; it interacts specifically with the DDX6 helicase Xp54; it is detected in polysomes and stalled translation initiation complexes; its over-expression leads to selective binding to translatable mRNA species without evidence of translation repression or mRNA degradation. Since both Xp54 and xRAPA are repressors of translation, activation appears to be effected through targeting of xRAPB/Xp54

Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery

Copyright @ 2014 Booth et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.When the nucleolus disassembles during open mitosis, many nucleolar proteins and RNAs associate with chromosomes, establishing a perichromosomal compartment coating the chromosome periphery. At present nothing is known about the function of this poorly characterised compartment. In this study, we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells. Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin. Following siRNA depletion of Ki-67, NIFK, B23, nucleolin, and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes. Correlative light and electron microscopy (CLEM) images suggest a near-complete loss of the entire perichromosomal compartment. Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells

The YY1 transcription factor is a component of ribonucleoprotein complexes in xenopus laevis oocytes and embryos.

Yin Yang 1 (YY1) is a multifunctional transcription factor that is known mainly for its ability to activate or initiate transcription of a wide assortment of genes involved in cellular growth and differentiation. Xenopus laevis oocytes and embryos were used as a model to identify and characterize a potential developmental role for YY1. Northern and Western blots of oocyte and embryonic extracts showed YY1 mRNA and protein is expressed from the earliest stages of oocyte development through to tadpole stages. Examination of the transcriptional activity of YY1 in both oocytes and embryos using reporter gene constructs containing YY1-binding elements demonstrated that YY1 does not act as a repressor or activator of transcription either in oocytes or in embryos. Sub-cellular fractionation of oocytes and Western blot analysis showed YY1 is localized almost exclusively to the cytoplasm of oocytes and in cells of early embryos. Sequence analysis of YY1 revealed that it contains an established RNA binding motif located within the zinc fingers. A series of biochemical assays were performed to address the possibility that YY1 functions as a component of mRNPs in the oocyte cytoplasm. RNA gel mobility shift analyses using in vitro synthesized histone H2A transcripts and supershifts using YY1-specific antibodies suggested that YY1 or YY1-containing complexes in cytoplasmic extracts were able to bind RNA. Chromatographic analysis of oocyte lysates showed YY1 was specifically retained on oligo (dT) cellulose columns. Treatment of the same lysates with RNase abolished binding to oligo (dT), indicating that retention is dependent on the presence of intact polyadenylated RNAs. This suggested that YY1 may be a component of messenger ribonucleoprotein particles (mRNP). Separation of oocyte lysates by size exclusion chromatography (SEC) revealed that YY1 was present in large complexes with an approximate molecular mass of 480 kDa. RNase or phosphatase treatment of oocyte extracts released YY1 from high mass complexes. Analysis of phosphatase or RNase-treated extracts for DNA binding activity showed that monomeric YY1 was able to bind DNA with high affinity. Immunoprecipitation of YY1 complexes followed by cDNA synthesis and sequencing revealed that YY1 is associated with both ribosomal and messenger RNAs in the cytoplasm of the oocyte. These results indicate a novel function for YY1 as a component of messenger ribonucleoprotein particles

Drosophila gene for antizyme requires ribosomal frameshifting for expression and contains an intronic gene for snRNP Sm D3 on the opposite strand

Journal ArticlePreviously, a Drosophila melanogaster sequence with high homology to the sequence for mammalian antizyme (ornithine decarboxylase antizyme) was reported. The present study shows that homology of this coding sequence to its mammalian antizyme counterpart also extends to a 5* open reading frame (ORF) which encodes the amino-terminal part of antizyme and overlaps the 11 frame (ORF2) that encodes the carboxy-terminal three-quarters of the protein. Ribosomes shift frame from the 5* ORF to ORF2 with an efficiency regulated by polyamines. At least in mammals, this is part of an autoregulatory circuit. The shift site and 23 of 25 of the flanking nucleotides which are likely important for efficient frameshifting are identical to their mammalian homologs. In the reverse orientation, within one of the introns of the Drosophila antizyme gene, the gene for snRNP Sm D3 is located. Previously, it was shown that two closely linked P-element transposon insertions caused the gutfeeling phenotype of embryonic lethality and aberrant neuronal and muscle cell differentiation. The present work shows that defects in either snRNP Sm D3 or antizyme, or both, are likely causes of the phenotype