NVL2 is a nucleolar AAA-ATPase that interacts with ribosomal protein L5 through its nucleolar localization sequence

NVL (nuclear VCP-like protein), a member of the AAA-ATPase family, is known to exist in two forms with N-terminal extensions of different lengths in mammalian cells. Here, we show that they are localized differently in the nucleus; NVL2, the major species, is mainly present in the nucleolus, whereas NVL1 is nucleoplasmic. Mutational analysis demonstrated the presence of two nuclear localization signals in NVL2, one of which is shared with NVL1. In addition, a nucleolar localization signal was found to exist in the N-terminal extra region of NVL2. The nucleolar localization signal is critical for interaction with ribosomal protein L5, which was identified as a specific interaction partner of NVL2 on yeast two-hybrid screening. The interaction of NVL2 with L5 is ATP-dependent and likely contributes to the nucleolar translocation of NVL2. The physiological implication of this interaction was suggested by the finding that a dominant negative NVL2 mutant inhibits ribosome biosynthesis, which is known to take place in the nucleolus.     

G-quadruplex DNA binding by a series of carbocyanine dyes.

We have examined a number of carbocyanine dyes for their ability to bind intramolecular G-quadruplex DNA structures (G4'-DNA) using a Taq polymerase stop assay. Of the five dyes examined, only one, N,N'-diethylthiacarbocyanine iodide (DTC), was found to bind to G4'-DNA. DTC was also the only dye found to inhibit human telomerase at 50 microM concentration.     

Perylene diimide G-quadruplex DNA binding selectivity is mediated by ligand aggregation

Two N,N'-disubstituted perylene diimide G-quadruplex DNA ligands, PIPER [N,N'-bis-(2-(1-piperidino)ethyl)-3,4,9,10-perylene tetracarboxylic acid diimide] and Tel01 [N,N'-bis-(3-(4-morpholino)-propyl)-3,4,9,10-perylene tetracarboxylic acid diimide] were studied. Visible absorbance, resonance light scattering, and fluorescence spectroscopy were used to characterize the pH-dependent aggregation of these ligands. The G-quadruplex DNA binding selectivity of these ligands as monitored by absorption spectroscopy is also pH-dependent. The ligands bind to both duplex and G-quadruplex DNA under low pH conditions, where the ligands are not aggregated. At higher pH, where the ligands are extensively aggregated, the apparent G-quadruplex DNA binding selectivity is high.     

Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, epsilon N182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant alphadelta site. Studies of the analogous mutation in the delta subunit, deltaN187Y, disclose rate constants for ACh occupancy of the nonmutant alpha epsilon site. The second CMS mutation, epsilon D175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. epsilon D175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, epsilon N182Y localizes to the interface with the alpha subunit, and epsilon D175 to the entrance of the ACh binding cavity. Both epsilon N182Y and epsilon D175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring epsilon N182 and epsilon D175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.     

Stimulation of ribosomal frameshifting by RNA G-quadruplex structures

Guanine-rich sequences can fold into four-stranded structures of stacked guanine-tetrads, so-called G-quadruplexes (G4). These unique motifs have been extensively studied on the DNA level; however, exploration of the biological roles of G4s at the RNA level is just emerging. Here we show that G4 RNA when introduced within coding regions are capable of stimulating −1 ribosomal frameshifting (−1 FS) in vitro and in cultured cells. Systematic manipulation of the loop length between each G-tract revealed that the −1 FS efficiency positively correlates with G4 stability. Addition of a G4-stabilizing ligand, PhenDC3, resulted in higher −1 FS. Further, we demonstrated that the G4s can stimulate +1 FS and stop codon readthrough as well. These results suggest a potentially novel translational gene regulation mechanism mediated by G4 RNA.     

Charge versus sequence for nuclear/nucleolar localization of plant ribosomal proteins

Ribosomal subunit assembly in the nucleolus is dependent on efficient targeting of ribosomal proteins (RPs) from the cytoplasm into the nucleus and nucleolus. Nuclear/nucleolar localization of a protein is generally mediated by one or more specific stretches of basic amino acids—nuclear/nucleolar localization signals (NLSs/NoLSs). Arabidopsis thaliana RPL23aA has eight putative NLSs/NoLSs (pNLSs/NoLSs). Here we mutated all eight NLS/NoLSs individually and in groups and showed, via transient expression in tobacco cells that nucleolar localization of RPL23aA was disrupted by mutation of various combinations of five or more pNLSs/NoLSs. Mutation of all eight pNLSs/NoLSs, a 50 % reduction in total basic charge of RPL23aA, resulted in a complete disruption of nucleolar localization, however, the protein can still localize to the nucleus. As no individual or specific combination of NoLSs was absolutely required for nucleolar localization, we suggest that nucleolar localization/retention of RPL23aA is dependent on the overall basic charge. In addition to the optimal basic charge conferred by these NoLSs, nucleolar localization/retention of RPL23aA also required a C-terminal putative 26S rRNA binding site. In contrast, in the RPs RPS8A and RPL15A, mutation of just two and three N-terminal pNLSs, respectively, disrupted both nuclear and nucleolar localization of these two RPs, indicating differential signal requirements for nuclear and nucleolar localization of the three Arabidopsis RPs RPL23aA, RPL15A and RPS8A.     

Synergic Role of Nucleophosmin Three-helix Bundle and a Flanking Unstructured Tail in the Interaction with G-quadruplex DNA

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control, and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold through multiple and transient electrostatic interactions, significantly enlarging the contact surface.     

An assay for human telomeric G-quadruplex DNA binding drugs

Compounds that stabilize the G-quadruplexes formed by human telomeres can inhibit the telomerase activity and are potential cancer therapies. We have developed an assay for the screening of compounds with high affinity for human telomeric G-quadruplexes (HTG). The assay uses a thiazole orange fluorescent reporter molecule conjugated to the aminoglycoside, neomycin, as a probe in a fluorescence displacement assay. The conjugation of the planar base stacking thiazole orange with the groove binding neomycin results in high affinity probe that can determine the relative binding affinity of high affinity HTG binding drugs in a high throughput format. The robust assay is applicable for the determination of the binding affinity of HTG in the presence of K⁺ or Na⁺.     

Anucleolate frog embryos contain ribosomal DNA sequences and a nucleolar antigen

Previous studies of Xenopus laevis embryos homozygous for the nucleolar deletion mutation have concluded that these embryos contain few, if any, copies of the genes for the 18 S and 28 S ribosomal RNAs. Using hybridization to restriction endonuclease digests of DNA it is found, in fact, that a small amount of ribosomal DNA is still present in such embryos. The ribosomal DNA in these embryos appears to include a few normal repeats together with a variety of unusual fragments containing either spacer or gene sequences. An antibody found in the serum of a scleroderma patient reacts with an antigen localized in the nucleoli of wild-type embryos. In anucleolate embryos this antigen is found in the so-called pseudonucleoli and in many small bodies in the nuclei.     

Nucleotide binding by the Mdm2 RING domain facilitates Arf-independent Mdm2 nucleolar localization

The RING domain of Mdm2 contains a conserved Walker A or P loop motif that is a characteristic of nucleotide binding proteins. We found that Mdm2 binds adenine-containing nucleotides preferentially and that nucleotide binding leads to a conformational change in the Mdm2 C terminus. Although nucleotide binding is not required for Mdm2 E3 ubiquitin ligase activity, we show that nucleotide binding-defective P loop mutants are impaired in p14(ARF)-independent nucleolar localization both in vivo and in vitro. Consistent with this, ATP-bound Mdm2 is preferentially localized to the nucleolus. Indeed, we identify a unique amino acid substitution in the P loop motif (K454A) that uncouples nucleolar localization and E3 ubiquitin ligase activity of Mdm2 and leads to upregulation of the E3 activity both in human cells and in Caenorhabditis elegans. We propose that nucleotide binding-facilitated nucleolar localization of Mdm2 is an evolutionarily conserved regulator of Mdm2 activity.     

Structural competition involving G-quadruplex DNA and its complement

Structural competition between the G-quadruplex, the I-motif, and the Watson-Crick duplex has been implicated for repetitive DNA sequences, but the competitive mechanism of these multistranded structures still needs to be elucidated. We investigated the effects of sequence context, cation species, and pH on duplex formation by the G-quadruplex of dG(3)(T(2)AG(3))(3) and its complement the I-motif of d(C(3)TA(2))(3)C(3), using ITC, DSC, PAGE, CD, UV, and CD stopped-flow kinetic techniques. ITC and PAGE experiments confirmed Watson-Crick duplex formation by the complementary strands. The binding constant of the two DNA strands in the presence of 10 mM Mg(2+) at pH 7.0 was shown to be 5.28 x 10(7) M(-1) at 20 degrees C, about 400 times larger than that in the presence of 100 mM Na(+) at pH 5.5. The dynamic transition traces of the duplex formation from the equimolar mixture of G-/C-rich complementary sequences were obtained at both pH 7.0 and pH 5.5. Fitting to a single-exponential function gave an observed rate of 8.06 x 10(-3) s(-1) at 20 degrees C in 10 mM Mg(2+) buffer at pH 7.0, which was about 10 times the observed rate at pH 5.5 under the same conditions. Both of the observed rates increased as temperature rose, implying that the dissociation of the single-stranded structured DNAs is the rate-limiting step for the WC duplex formation. The difference between the apparent activation energy at pH 7.0 and that at pH 5.5 reflects the fact that pH significantly influences the structural competition between the G-quadruplex, the I-motif, and the Watson-Crick duplex, which also implies a possible biological role for I-motifs in biological regulation.     

One-step synthesis of methylene-bridged bis-carbazole and evaluation of its antitumor activity and G-quadruplex DNA binding property

Most reported carbazolyl G-quadruplex DNA (G4-DNA) ligands possess a rigid structure rather than a flexible one. The conformationally flexible ligands are paid much less attention. In this study, we report a novel class of non-rigid methylene-bridged biscarbazolyl ligand and their G4-DNA binding properties. Moreover, the antitumor activities of all these oligomers have been evaluated. The results show that this family of oligomers could be facilely synthesized via solely one step. Among them, compound 2, the bis-carbazole derivative, displays the best antitumor activity and IC₅₀ values against HT-29, HepG2, A375 and MCF-7 cells are 0.69, 5.09, 3.15 and 3.8 μ mol/L, respectively. Although conformationally flexible, 2 is still capable of binding to as well as stabilizing G4-DNA via π-π stacking interaction. Moreover, 2 selectively binds to G4-DNA over duplex DNA. The current study enriches the category of carbazolyl G4-DNA ligands and paves the way for the search of more efficient G4-DNA ligands and antitumor leads.     

DNA binding properties of a 110 kDa nucleolar protein.

A single strand specific DNA binding protein was purified to homogeneity from calf thymus nucleoprotein. The monomeric protein is elongated in shape and has a molecular mass of 110 kDa. Since immunocytochemistry revealed that the protein is predominantly located in the nucleolus we refer to it as the 110 kDa nucleolar protein. The protein binds not only to single stranded DNA but also to single stranded RNA, including homopolymeric synthetic RNA. We have used the single stranded DNA binding properties of the 110 kDa protein in model studies to investigate its effects on the configuration of nucleic acid. Our results are: only 50-55 protein molecules are sufficient to saturate all binding sites on the 6408 nucleotides of phage fd DNA; protein binding cause a compaction of single stranded DNA; large nucleoprotein aggregates are formed in the presence of divalent cations; this is due to protein-protein interactions which occur at moderately high concentrations of magnesium-, calcium or manganese ions; the protein induces the reassociation of complementary nucleic acid sequences. We speculate that the 110 kDa protein performs similar reactions in vivo and may have a function related to the processing and packaging of preribosomal RNA.     

Nucleolar localization of parafibromin is mediated by three nucleolar localization signals

Parafibromin is a putative tumor suppressor encoded by HRPT2 and implicated in parathyroid tumorigenesis. We previously reported a functional bipartite nuclear localization signal (NLS) at residues 125-139. We now demonstrate that parafibromin exhibits nucleolar localization, mediated by three nucleolar localization signals (NoLS) at resides 76-92, 192-194 and 393-409. These NoLS represent clusters of basic amino acids arginine and lysine, similar to those found in other nucleolar proteins, as well as being characteristic of NLSs. While parafibromin's bipartite NLS is the primary determinant of nuclear localization, it does not mediate nucleolar localization. In contrast, the three identified NoLSs play only a minor role in nuclear localization, but are critical for the nucleolar localization of parafibromin.     

Extension of G-quadruplex DNA by ciliate telomerase

Telomeric DNA can fold into four-stranded structures known as G-quadruplexes. Here we investigate the ability of G-quadruplex DNA to serve as a substrate for recombinant Tetrahymena and native Euplotes telomerase. Inter- and intramolecular G-quadruplexes were gel-purified and their stability examined using native gel electrophoresis, circular dichroism (CD) and thermal denaturation. While intermolecular G-quadruplexes were highly stable, they were excellent substrates for both ciliate telomerases in primer extension assays. In contrast, intramolecular G-quadruplexes formed in K+ exhibited biphasic unfolding and were not extended by ciliate telomerases. Na+-stabilised intramolecular G-quadruplexes were extended by telomerase owing to their rapid rate of dissociation. The Tetrahymena telomerase protein component bound to inter- but not intramolecular K+-stabilised G-quadruplexes. This study provides evidence that parallel intermolecular G-quadruplexes can serve as substrates for telomerase in vitro, their extension being mediated through direct interactions between this higher-order structure and telomerase.