Mammalian nucleostemin (NS) is a nucleolar guanosine triphosphate-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1-4). NS1 is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed green fluorescent protein (GFP)-NS1 enrich within the nucleolar granular regions as expected, whereas overexpressed GFP-NS1 localized throughout the nucleolus and nucleoplasm, and to several transcriptionally active interbands of polytene chromosomes. Severe overexpression correlated with the appearance of melanotic tumors and larval/pupal lethality. Depletion of 60% of NS1 transcripts also lead to larval and pupal lethality. NS1 protein depletion correlated with the loss of imaginal island (precursor) cells in the larval midgut and to an apparent block in the nucleolar release of large ribosomal subunits in terminally differentiated larval midgut polyploid cells. Ultrastructural examination of larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes and a concomitant appearance of cytoplasmic preautophagosomes and lysosomes. We interpret the appearance of these structures as indicators of cell stress response. INTRODUCTIONMammalian nucleostemin (NS) is a nucleolar guanosine triphosphate (GTP)-binding protein first characterized in embryonic and neuronal stem cells and in certain cancer cells where it probably plays regulatory roles in cell cycle progression and ribosome biogenesis McKay, 2002, 2005; reviewed by Ma and Pederson, 2008b). Steadystate concentrations of NS drop to undetectable levels just before rat cortical stem cell differentiation (Tsai and McKay, 2002), suggesting that reduced expression of NS regulates stem cell proliferation and differentiation by triggering exit from the cell cycle (Normile, 2002;Tsai and McKay, 2002;Beekman et al., 2006). Mammalian NS rapidly cycles between the nucleolus and the nucleoplasm. Retention of NS within the nucleolus is prolonged when it binds GTP (Misteli, 2005;Tsai and McKay, 2005;Meng et al., 2006Meng et al., , 2007. GTP bound to the central region of NS is thought to stabilize interactions between its amino-terminal basic domain and other nucleolar components, whereas NS redistributes to the nucleoplasm when GTP binding is impaired by mutation (Tsai and McKay, 2005). Coimmunoprecipitation, yeast twohybrid, and bimolecular fluorescence complementation assays demonstrated an interaction between NS and nucleophosmin (B23) (Ma and Pederson, 2008a), a multifunctional chaperone that probably participates in the later stages of ribosome assembly within the granular component of nucleoli. Similar to NS, nucleophosmin probably plays a role in cell proliferation (Szebeni et al., 2003;Grisendi et al., 2006). Recent studies suggest that NS ...
Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of structural damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this paper, we describe the application of MALDI-TOF-MS to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with non-standard synthesis, deprotection or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and utilization.
Nopp140 associates with small nucleolar RNPs to chaperone pre-rRNA processing and ribosome assembly. Alternative splicing yields two isoforms in Drosophila: Nopp140-True is homologous to vertebrate Nopp140 particularly in its carboxy terminus, whereas Nopp140-RGG contains a glycine and arginine-rich (RGG) carboxy terminus typically found in vertebrate nucleolin. Loss of ribosome function or production at critical points in development leads to Minute phenotypes in Drosophila or the Treacher Collins syndrome (TCS) in humans. To ascertain the functional significance of Nopp140 in Drosophila development, we expressed interfering RNA using the GAL4/UAS system. Reverse transcription-PCR showed variable losses of Nopp140 mRNA in larvae from separate RNAi-expressing transgenic lines, whereas immunofluorescence microscopy with isoform-specific antibodies showed losses of Nopp140 in imaginal and polyploid tissues. Phenotypic expression correlated with the percent loss of Nopp140 transcripts: a >50% loss correlated with larval and pupal lethality, disrupted nuclear structures, and in some cases melanotic tumors, whereas a 30% loss correlated with adult wing, leg, and tergite deformities. We consider these adult phenotypes to be Minute-like and reminiscent of human craniofacial malformations associated with TCS. Similarly, overexpression of either isoform caused embryonic and larval lethality, thus indicating proper expression of Nopp140 is critical for normal development. INTRODUCTIONNucleolar phosphoprotein 140 (Nopp140, M r ϭ 140 -180 kDa) has several purported functions in ribosome assembly (Meier, 2005). The greatest steady state concentrations of vertebrate Nopp140 occur within the dense fibrillar component of nucleoli and within extra-nucleolar Cajal bodies. Nopp140 associates with box C/D small nucleolar ribonucleoprotein (snoRNPs) that guide site-specific 2Ј-O-methylation of the pre-rRNA and with box H/ACA snoRNPs that guide site-specific pseudouridylation of pre-rRNA and snRNAs (Yang et al., 2000;Wang et al., 2002; reviewed by Meier, 2005). Nopp140 likely acts as an assembly factor for these snoRNPs or as a chaperone in their transport between Cajal bodies and nucleoli (Isaac et al., 1998;Lemm et al., 2006). Nopp140 also interacts with RNA Pol I to regulate rRNA transcription within mammalian nucleoli (Chen et al., 1999) and with Pol II transcription factors in the protein kinase A-mediated activation of the ␣ 1 -acid glycoprotein gene in liver cells during acute phase response (Miau et al., 1997;Chiu et al., 2002). Nopp140 shuttles rapidly between the nucleus and the cytoplasm (Meier and Blobel, 1992), perhaps to facilitate the import of nucleolar ribosome assembly factors or the export of nucleolar products (Meier and Blobel, 1990).In Drosophila melanogaster, the Nopp140 gene maps to the proximal left arm of chromosome 3 in cytological region 78F4 (http://flybase.bio.indiana.edu/.bin/fbidq.html? FBgn0037137). Two isoforms of the protein exist: Nopp140-True contains 686 amino acid residues and Nopp140-RGG conta...
DNAzyme-based biosensor can complement enzyme-based media to identify E. coli rapidly.
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