scholarly journals A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

2000 ◽  
Vol 11 (4) ◽  
pp. 1153-1167 ◽  
Author(s):  
Rudolf F. Zirwes ◽  
Jens Eilbracht ◽  
Sandra Kneissel ◽  
Marion S. Schmidt-Zachmann
Keyword(s):  
Cultured Cells ◽  
Gradient Centrifugation ◽  
Intracellular Localization ◽  
Ribosomal Subunit ◽  
Rnase A ◽  
Rna Helicases ◽  
Dead Box ◽  
Transcription Inhibitor ◽  
Calculated Mass

We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of “DEAD-box” proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with ∼11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with ∼4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit.

scholarly journals Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cells.

1984 ◽  
Vol 99 (2) ◽  
pp. 471-480 ◽  
Author(s):  
K J Ibbotson ◽  
G D Roodman ◽  
L M McManus ◽  
G R Mundy
Keyword(s):  
Bone Resorption ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Gradient Centrifugation ◽  
Cell Formation ◽  
Tartrate Resistant Acid Phosphatase ◽  
Nonspecific Esterase ◽  
Multinucleated Cells

The predominant cell responsible for bone resorption, the multinucleated osteoclast, has been difficult to study because of inaccessibility. When feline marrow-derived mononuclear cells are established in long-term culture, multinucleated cells form within 48 h, reaching maximum numbers at 16 d. We have observed that these cultured cells have many of the features of osteoclasts. Morphologically, they are multinucleated, contain large numbers of branched mitochondria, have a peripheral cytoplasm lacking organelles (a clear zone), and have extensive cell-surface processes. In addition to these ultrastructural features, the cells contain a tartrate-resistant acid phosphatase, the activity of which is increased by parathyroid hormone (PTH) and inhibited by calcitonin. PTH, prostaglandin E2, and 1,25(OH)2 vitamin D3 increased multinucleated cell formation, while calcitonin inhibited the stimulatory effects of PTH. Time-lapse cinemicrographic and autoradiographic studies indicated that the multinucleated cells formed by fusion of the mononuclear progenitors. The multinucleated cells were phagocytic and stained with nonspecific esterase, consistent with their being derived from immature monocytes. Further, cell populations enriched for multinucleated cells release 45Ca from devitalized bone. Density-gradient centrifugation on Percoll was used to enrich and characterize the mononuclear progenitors of these multinucleated cells. The progenitor cells were found predominantly in Percoll density layers of 1.065 to 1.08 g/ml and were enriched up to 30-fold as compared to unfractionated cells. The bone marrow mononuclear cells that formed the multinucleated cells were initially nonadherent to plastic, stained heavily with nonspecific esterase, and appeared to be immature monocytes histologically. These data indicate that the multinucleated osteoclast-like cells in our cultures are derived from nonadherent monocytic progenitor cells that are responsive to osteotropic hormones. The ability to grow and characterize these cells in vitro should facilitate studies to elucidate the role these cells play in normal and pathologic states of bone resorption.

scholarly journals Metabolism and intracellular localization of a fluorescently labeled intermediate in lipid biosynthesis within cultured fibroblasts.

1981 ◽  
Vol 91 (3) ◽  
pp. 872-877 ◽  
Author(s):  
R E Pagano ◽  
K J Longmuir ◽  
O C Martin ◽  
D K Struck
Keyword(s):  
Endoplasmic Reticulum ◽  
Phosphatidic Acid ◽  
Cultured Cells ◽  
Large Fraction ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Cultured Fibroblasts ◽  
Acid Analogue ◽  
Unique Method

In this paper we report on the uptake and distribution of an exogenously supplied fluorescent phosphatidic acid analogue by Chinese hamster fibroblasts. Under appropriate in vitro incubation conditions, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidic acid was rapidly and preferentially transferred from phospholipid vesicles to cells at 2 degrees C. However, unlike similar fluorescent derivatives of phosphatidylcholine and phosphatidylethanolamine that remain restricted to the plasma membrane under such incubation conditions (Struck, D. K., and R. E. Pagano. 1080. J. Biol. Chem. 255:5405--5410), most of the phosphatidic acid-derived fluorescence was localized at the nuclear membrane, endoplasmic reticulum, and mitochondria. This was shown by labeling cells with rhodamine-containing probes specific for mitochondria or endoplasmic reticulum, and comparing the patterns of intracellular NBD and rhodamine fluorescence. Extraction and analysis of the fluorescent lipids associated with the cells after treatment with vesicles at 2 degrees or 37 degrees C revealed that a large fraction of the fluorescent phosphatidic acid was converted to fluorescent diglyceride, phosphatidylcholine, and triglyceride. Our findings suggest that fluorescent phosphatidic acid may be useful in correlating biochemical studies of lipid metabolism in cultured cells and studies of the Intracellular localization of the metabolites by fluorescence microscopy. In addition, this compound provides a unique method for visualizing the endoplasmic reticulum in living cells.

scholarly journals A Role for DEAD Box 1 at DNA Double-Strand Breaks

2008 ◽  
Vol 28 (20) ◽  
pp. 6413-6425 ◽  
Author(s):  
Lei Li ◽  
Elizabeth A. Monckton ◽  
Roseline Godbout
Keyword(s):  
Ionizing Radiation ◽  
Neuroblastoma Cell ◽  
Rnase H ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Rna Helicases ◽  
Strand Breaks ◽  
Dead Box

ABSTRACT DEAD box proteins are a family of putative RNA helicases associated with all aspects of cellular metabolism involving the modification of RNA secondary structure. DDX1 is a member of the DEAD box protein family that is overexpressed in a subset of retinoblastoma and neuroblastoma cell lines and tumors. DDX1 is found primarily in the nucleus, where it forms two to four large aggregates called DDX1 bodies. Here, we report a rapid redistribution of DDX1 in cells exposed to ionizing radiation, resulting in the formation of numerous foci that colocalize with γ-H2AX and phosphorylated ATM foci at sites of DNA double-strand breaks (DSBs). The formation of DDX1 ionizing-radiation-induced foci (IRIF) is dependent on ATM, which was shown to phosphorylate DDX1 both in vitro and in vivo. The treatment of cells with RNase H prevented the formation of DDX1 IRIF, suggesting that DDX1 is recruited to sites of DNA damage containing RNA-DNA structures. We have shown that DDX1 has RNase activity toward single-stranded RNA, as well as ADP-dependent RNA-DNA- and RNA-RNA-unwinding activities. We propose that DDX1 plays an RNA clearance role at DSB sites, thereby facilitating the template-guided repair of transcriptionally active regions of the genome.

scholarly journals Analyses of the Functional Regions of DEAD-Box RNA “Helicases” with Deletion and Chimera Constructs Tested In Vivo and In Vitro

2011 ◽  
Vol 413 (2) ◽  
pp. 451-472 ◽  
Author(s):  
Josette Banroques ◽  
Olivier Cordin ◽  
Monique Doère ◽  
Patrick Linder ◽  
N. Kyle Tanner
Keyword(s):  
Rna Helicases ◽  
Functional Regions ◽  
Dead Box

scholarly journals Regulation of Alternative Splicing by the ATP-Dependent DEAD-Box RNA Helicase p72

2002 ◽  
Vol 22 (16) ◽  
pp. 5698-5707 ◽  
Author(s):  
Arnd Hönig ◽  
Didier Auboeuf ◽  
Marjorie M. Parker ◽  
Bert W. O'Malley ◽  
Susan M. Berget
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Globin Gene ◽  
Rna Helicase ◽  
Close Relative ◽  
Rna Helicases ◽  
Enhancer Element ◽  
Dead Box ◽  
Precursor Rna

ABSTRACT Although a number of ATP-dependent RNA helicases are important for constitutive RNA splicing, no helicases have been implicated in alternative RNA splicing. Here, we show that the abundant DEAD-box RNA helicase p72, but not its close relative p68, affects the splicing of alternative exons containing AC-rich exon enhancer elements. The effect of p72 was tested by using mini-genes that undergo different types of alternative splicing. When the concentration of p72 was increased in transient transfections, the inclusion of enhancer-containing CD44 alternative exons v4 and v5 increased using a mini-gene that contained these exons and their flanking introns inserted into a β-globin gene. Other types of alternative splicing were not impacted by altering p72 concentrations. Mutation of the p72 helicase ATP-binding site or deletion of the carboxy-terminal region of the protein reduced the ability of the transfected protein to affect CD44 variable exon splicing. Use of in vitro extracts overexpressing p72 indicated that p72 becomes associated with complexes containing precursor RNA. Helicases have been implicated both in altering RNA-RNA interactions and in remodeling RNA-protein complexes. CD44 exon v4 contains a potential internal secondary structure element that base pairs the 5′ splice site with a region inside the exon located between enhancer elements. Mutations that destroyed this complementarity modestly increased inclusion in the absence of p72 but still responded to increasing p72 concentration like the wild-type exon, suggesting that p72 might have effects on protein-RNA interactions. In agreement with this hypothesis, p72 was not able to restore the inclusion of an exon mutated for its major enhancer element. Our results suggest that RNA helicases may be important alternative splicing regulatory factors.

scholarly journals Dead or alive: DEAD-box ATPases as regulators of ribonucleoprotein complex condensation

2021 ◽  
Vol 402 (5) ◽  
pp. 653-661
Author(s):  
Karsten Weis
Keyword(s):  
Rna Processing ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Dependent Manner ◽  
Rna Helicases ◽  
Ribonucleoprotein Complexes ◽  
Dead Box ◽  
Rna Duplexes ◽  
Rna Protein Complexes

Abstract DEAD-box ATPase proteins are found in all clades of life and have been associated with a diverse array of RNA-processing reactions in eukaryotes, bacteria and archaea. Their highly conserved core enables them to bind RNA, often in an ATP-dependent manner. In the course of the ATP hydrolysis cycle, they undergo conformational rearrangements, which enable them to unwind short RNA duplexes or remodel RNA-protein complexes. Thus, they can function as RNA helicases or chaperones. However, when their conformation is locked, they can also clamp RNA and create ATP-dependent platforms for the formation of higher-order ribonucleoprotein complexes. Recently, it was shown that DEAD-box ATPases globally regulate the phase-separation behavior of RNA-protein complexes in vitro and control the dynamics of RNA-containing membraneless organelles in both pro- and eukaryotic cells. A role of these enzymes as regulators of RNA-protein condensates, or ‘condensases’, suggests a unifying view of how the biochemical activities of DEAD-box ATPases are used to keep cellular condensates dynamic and ‘alive’, and how they regulate the composition and fate of ribonucleoprotein complexes in different RNA processing steps.

scholarly journals Gene Therapeutic Approach for Inhibiting Hepatitis C Virus Replication Using a Recombinant Protein That Controls Interferon Expression

2010 ◽  
Vol 54 (12) ◽  
pp. 5048-5056 ◽  
Author(s):  
Chul Hyun Joo ◽  
Uk Lee ◽  
Young Ran Nam ◽  
Jae U. Jung ◽  
Heuiran Lee ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cultured Cells ◽  
Viral Vector ◽  
Wide Spectrum ◽  
Intracellular Localization ◽  
Treatment Modalities ◽  
In Cells

ABSTRACT The hepatitis C virus (HCV) is a continuing threat to public health. The systemic administration of interferon alpha with ribavirin is the only currently approved treatment. However, this treatment is associated with a wide spectrum of systemic side effects that limits its effectiveness; thus, there is an urgent need for new treatment modalities. In this study, we describe a novel anti-HCV strategy employing a recombinant transcription factor that we have engineered in such a way that NS3/4a viral protease controls its intracellular localization, thereby restoring interferon secretion specifically in cells infected with HCV. Proof-of-concept experiments validated the strategy, showing that the recombinant transcription factor was triggered to stimulate interferon promoter by NS3/4A and remained inactive in cells without NS3/4a. Using an adenovirus-associated viral vector delivery system, we found that the recombinant transcription factor inhibited HCV replication effectively in vitro in cultured cells.

scholarly journals ALG-2-interacting Tubby-like protein superfamily member PLSCR3 is secreted by an exosomal pathway and taken up by recipient cultured cells

2013 ◽  
Vol 33 (2) ◽  
Author(s):  
Tatsutoshi Inuzuka ◽  
Akira Inokawa ◽  
Cen Chen ◽  
Kumiko Kizu ◽  
Hiroshi Narita ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Gradient Centrifugation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Established Cell Line ◽  
Sucrose Density Gradient Centrifugation ◽  
Hek 293 ◽  
Aaa Atpase

PLSCRs (phospholipid scramblases) are palmitoylated membrane-associating proteins. Regardless of the given names, their physiological functions are not clear and thought to be unrelated to phospholipid scrambling activities observed in vitro. Using a previously established cell line of HEK-293 (human embryonic kidney-293) cells constitutively expressing human Scr3 (PLSCR3) that interacts with ALG-2 (apoptosis-linked gene 2) Ca2+-dependently, we found that Scr3 was secreted into the culture medium. Secretion of Scr3 was suppressed by 2-BP (2-bromopalmitate, a palmitoylation inhibitor) and by GW4869 (an inhibitor of ceramide synthesis). Secreted Scr3 was recovered in exosomal fractions by sucrose density gradient centrifugation. Palmitoylation sites and the N-terminal Pro-rich region were necessary for efficient secretion, but ABSs (ALG-2-binding sites) were dispensable. Overexpression of GFP (green fluorescent protein)-fused VPS4BE235Q, a dominant negative mutant of an AAA (ATPase associated with various cellular activities) ATPase with a defect in disassembling ESCRT (endosomal sorting complex required for transport)-III subunits, significantly reduced secretion of Scr3. Immunofluorescence microscopic analyses showed that Scr3 was largely localized to enlarged endosomes induced by overexpression of a GFP-fused constitutive active mutant of Rab5A (GFP–Rab5AQ79L). Secreted Scr3 was taken up by HeLa cells, suggesting that Scr3 functions as a cell-to-cell transferable modulator carried by exosomes in a paracrine manner.

scholarly journals Qsr1p, a 60S ribosomal subunit protein, is required for joining of 40S and 60S subunits.

1997 ◽  
Vol 17 (9) ◽  
pp. 5136-5145 ◽  
Author(s):  
D P Eisinger ◽  
F A Dick ◽  
B L Trumpower
Keyword(s):  
Gradient Centrifugation ◽  
Ribosomal Subunit ◽  
Inducible Promoter ◽  
Temperature Sensitive ◽  
Subunit Protein ◽  
Temperature Sensitive Mutants ◽  
Ribosomal Subunit Protein ◽  
Subunit Joining ◽  
Mutant Forms

QSR1 is a recently discovered, essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein. Thirty-one unique temperature-sensitive alleles of QSR1 were generated by regional codon randomization within a conserved 20-amino-acid sequence of the QSR1-encoded protein. The temperature-sensitive mutants arrest as viable, large, unbudded cells 24 to 48 h after a shift to 37 degrees C. Polysome and ribosomal subunit analysis by velocity gradient centrifugation of lysates from temperature-sensitive qsr1 mutants and from cells in which Qsr1p was depleted by down regulation of an inducible promoter revealed the presence of half-mer polysomes and a large pool of free 60S subunits that lack Qsr1p. In vitro subunit-joining assays and analysis of a mutant conditional for the synthesis of Qsr1p demonstrate that 60S subunits devoid of Qsr1p are unable to join with 40S subunits whereas 60S subunits that contain either wild-type or mutant forms of the protein are capable of subunit joining. The defective 60S subunits result from a reduced association of mutant Qsr1p with 60S subunits. These results indicate that Qsr1p is required for ribosomal subunit joining.

scholarly journals The E1∧E4 Protein of Human Papillomavirus Type 16 Associates with a Putative RNA Helicase through Sequences in Its C Terminus

2000 ◽  
Vol 74 (21) ◽  
pp. 10081-10095 ◽  
Author(s):  
John Doorbar ◽  
Robert C. Elston ◽  
Sawsan Napthine ◽  
Kenneth Raj ◽  
Elizabeth Medcalf ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Cell Lines ◽  
Mrna Stability ◽  
Ribosome Biogenesis ◽  
Coat Proteins ◽  
Rna Helicases ◽  
Human Papillomavirus Type 16 ◽  
Dead Box ◽  
C Terminus

ABSTRACT Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1∧E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1∧E4.L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1∧E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1∧E4 (in vivo and in vitro) and could be immunoprecipitated as an E1∧E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1∧E4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1∧E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with threeEscherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1∧E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1∧E4-associated protein indicates a possible role for E1∧E4 in virus synthesis.

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