Fibrillization Process of Human Amyloid-Beta Protein (1-40) under a Molecular Crowding Environment Mimicking the Interior of Living Cells Using Cell Debris.

Molecular crowding environments play a crucial role in understanding the mechanisms of biological reactions. Inside living cells, a diverse array of molecules coexists within a volume fraction ranging from 10% to 30% v/v. However, conventional spectroscopic methods often face difficulties in selectively observing the structures of particular proteins or membranes within such molecularly crowded environments due to the presence of high background signals. Therefore, it is crucial to establish in vitro measurement conditions that closely resemble the intracellular environment. Meanwhile, the neutron scattering method offers a significant advantage in selectively observing target biological components, even within crowded environments. Recently, we have demonstrated a novel scattering method capable of selectively detecting the structures of targeted proteins or membranes in a closely mimicking intracellular milieu achieved utilizing whole-cell contents (deuterated-cell debris). This method relies on the inverse contrast matching technique in neutron scattering. By employing this method, we successfully observed the fibrillization process of human amyloid beta-protein (Aß 1-40) under a molecular crowding environment (13.1% w/v cell debris, Aß/cell debris = ~1/25 w/w) that closely mimics the interior of living cells. Aß protein is well known as a major pathogenic component of Alzheimer's disease. The present results combining model simulation analyses clearly show that the intracellular environment facilitates the potential formation of even more intricate higher-order aggregates of Aß proteins than those previously reported.

Magnetic field effects on the structure and molecular behavior of pigeon iron-sulfur protein.

Pigeon iron-sulfur (Fe-S) cluster assembly 1 homolog (clISCA1) is a target protein for research into the biomagnetoreception mechanism, as the clCRY4/clISCA1 oligomer, a complex composed of the columnar clISCA1 oligomer and the magnetosensor candidate protein cryptochrome-4 (clCRY4) oligomer, tends to orient itself along weak magnetic fields, such as geomagnetic fields, under blue light. To obtain insight into the magnetic orientation mechanism of the clCRY4/clISCA1 oligomer, we inspected magnetic field effects on the structure and molecular behavior of clISCA1 by small angle X-ray scattering analysis. The results indicated that the clISCA1 protomer took the Fe-S cluster-bound globular form and unbound rod-like form. The globular clISCA1 protomer assembled to form columnar oligomers, which allowed for the binding of many Fe-S clusters at the interface between clISCA1 protomers. Moreover, the translational diffusion and the columnar oligomerization of clISCA1 were controlled by the external static magnetic field and Fe-S clusters bound to clISCA1. However, the columnar clISCA1 oligomer was not oriented along the external static magnetic field (~1 T) when clCRY4 was not bound to clISCA1. This result indicated that clCRY4 has a function to enhance the magnetic orientational property of clCRY4/clISCA1 oligomer.

Expression and characterization of l-arabinose isomerase from Geobacillus stearothermophilus for improved activity under acidic condition.

A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions.

Catalytic mechanism and evolutionary characteristics of thioredoxin from Halobacterium salinarum NRC-1.

Thioredoxin (TRX) is an important antioxidant against oxidative stress. TRX from the extremely halophilic archaeon Halobacterium salinarum NRC-1 (HsTRX-A), which has the highest acidic residue content [(Asp + Glu)/(Arg + Lys + His) = 9.0] among known TRXs, was chosen to elucidate the catalytic mechanism and evolutionary characteristics associated with haloadaptation. X-ray crystallographic analysis revealed that the main-chain structure of HsTRX-A is similar to those of homologous TRXs; for example, the root-mean-square deviations on Cα atoms were <2.3 Šfor extant archaeal TRXs and <1.5 Šfor resurrected Precambrian TRXs. A unique water network was located near the active-site residues (Cys45 and Cys48) in HsTRX-A, which may enhance the proton transfer required for the reduction of substrates under a high-salt environment. The high density of negative charges on the molecular surface (3.6 × 10-3 e Å-2) should improve the solubility and haloadaptivity. Moreover, circular-dichroism measurements and enzymatic assays using a mutant HsTRX-A with deletion of the long flexible N-terminal region (Ala2-Pro17) revealed that Ala2-Pro17 improves the structural stability and the enzymatic activity of HsTRX-A under high-salt environments (>2 M NaCl). The elongation of the N-terminal region in HsTRX-A accompanies the increased hydrophilicity and acidic residue content but does not affect the structure of the active site. These observations offer insights into molecular evolution for haloadaptation and potential applications in halophilic protein-related biotechnology.

Expression, Folding, and Activation of Halophilic Alkaline Phosphatase in Non-Halophilic Brevibacillus choshinensis.

Halophilic enzymes contain a large number of acidic amino acids and marginal large hydrophobic amino acids, which make them highly soluble even under strongly hydrophobic conditions. This characteristic of halophilic enzymes provides potential for their industrial application. However, halophilic enzymes easily degrade when used for industrial applications compared with enzymes from other extremophiles because of their instability in low-salt environments. We aimed to clarify the stabilization mechanism of halophilic enzymes. We previously attempted to express halophilic alkaline phosphatase from Halomonas (HaALP) in non-halophilic E. coli. However, the expressed HaALP showed little activity. Therefore, we overexpressed HaALP in Gram-positive non-halophilic Brevibacillus choshinensis in this study, which was successfully expressed and purified in its active form. HaALP was denatured in 6 M urea, refolded using various salts and the non-ionic osmolyte trimethylamine N-oxide (TMAO), and assessed by native polyacrylamide gel electrophoresis. HaALP refolded in 3 M NaCl or 3 M TMAO containing Na+ ions. Hydrophobic interactions due to a high salt concentration or TMAO enhanced the formation of the folding intermediate (the monomer precursor), and only Na+ ions activated the dimer form. This insight into the stabilization mechanism of HaALP may lead to the development of industrial applications of halophilic enzymes under low-salt conditions.

Observation of Protein and Lipid Membrane Structures in a Model Mimicking the Molecular-Crowding Environment of Cells Using Neutron Scattering and Cell Debris.

The interior of living cells is a molecular-crowding environment, where large quantities of various molecules coexist. Investigations into the nature of this environment are essential for an understanding of both the elaborate biological reactions and the maintenance of homeostasis occurring therein. The equilibrium states of biological macromolecular systems are affected by molecular-crowding environments unmatched by in vitro diluted environments; knowledge about crowding effects is still insufficient due to lack of relevant experimental studies. Recent developments in the techniques of in-cell NMR and large-scale molecular dynamics simulation have provided new insights into the structure and dynamics of biological molecules inside the cells. This study focused on a new experimental technique to directly observe the structure of a specific protein or membrane in condensed crowder solutions using neutron scattering. Deuterated whole-cell debris was used to reproduce an environment that more closely mimics the interior of living cells than models used previously. By the reduction of the background scattering from large amounts of cell debris, we successfully extracted structure information for both small globular protein and small unilamellar vesicle (SUV) from the concentrated cell-debris solution up to a weight ratio of 1:60 for protein/crowder and 1:40 for SUV/crowder.

The non-glycosylated N-terminal domain of human thrombopoietin is a molten globule under native conditions.

Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1-163 residues of hTPO (hTPO163 ) including the N-terminal active site domain (1-153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO163 is higher than that of the full-length glycosylated hTPO (ghTPO332 ). In order to clarify the structural and physicochemical properties of hTPO163 , circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO163 undergoes an induced-fit conformational change (+19.0% for helix and -16.7% for ß-strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO163 is a multistate transition; hTPO163 is thermally stabilized upon receptor (c-Mpl) binding, as indicated with raising the midpoint (Tm ) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO163 indicate that hTPO163 exists as a molten globule under native conditions, which may enable the induced-fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C-terminal domain (154-332 residues) and glycosylation assists the folding of the N-terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO163 might be reduced, if the conformational variability of hTPO163 is restricted by mutation and/or by the addition of C-terminal domain with glycosylation to keep its conformation suitable for the c-Mpl recognition.

Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein facilitating the extreme radiation resistance of Deinococcus radiodurans. PprA is a unique protein to the genus Deinococcus and exists as an oligomer ranging from a tetramer to an ∼100-mer depending on protein concentrations. Here, the X-ray crystal structure of PprA was determined to clarify how PprA confers radiation resistance. The tertiary structure of dimeric PprA was elucidated by using mutants obtained with random and site-directed mutagenesis methods (W183R and A139R); these mutants have disabled DNA binding and polymerization functions. Because the mutant A139R and W183R proteins have dimeric assemblies with 2 different interfaces (Interfaces 1 and 2), the linear and oligomerized PprA model was constructed as a left-handed face-to-face periodic screw structure. In addition, the linear structure in solution was confirmed by small-angle scattering experiments. The site-directed mutational analysis identified essential basic amino acids for DNA binding. These analytical data support the hypothesis that a complex assembly of PprA molecules, which are extended and have a screw structure, surrounds and stretches the DNA strand, acting as a novel guide to colocalize the DNA strands for efficient DNA repairs.-Adachi, M., Shimizu, R., Shibazaki, C., Satoh, K., Fujiwara, S., Arai, S., Narumi, I., Kuroki, R. Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Improved substrate specificity for D-galactose of L-arabinose isomerase for industrial application.

L-Arabinose isomerase isolated from Geobacillus stearothermophilus (GSAI) was modified to improve its substrate specificity for D-galactose for the production of D-tagatose, a potential reduced-energy sweetener. Among the selected residues, mutation at residue 18 produced a mutant strain, H18T, which exhibited increased activity for D-galactose compared with the wild-type (WT) enzyme. Analysis of the substrate specificity of H18T showed a 45.4% improvement for D-galactose. Replacing histidine with threonine at residue 18 resulted in approximately 2.7-fold and 1.8-fold higher substrate binding and catalytic efficiency, respectively, for D-galactose. Further enhancement of the specific activity and catalytic efficiency of H18T for D-galactose by up to 2.7-fold and 4.3-fold, respectively, was achieved by adding borate during L-arabinose isomerase catalysis. Moreover, H18T showed thermostability and no destabilization was detected, which is promising for the industrial production of D-tagatose.

Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography.

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Šresolution) and X-ray (1.1 Šresolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%-75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.

Expression and characterization of the Renilla luciferase with the cumulative mutation.

Luciferase from Renilla reniformis (RLuc) is a good research tool as a reporter protein and bioimaging probes, yielding blue light using the substrate coelenterazine. However, the applications are limited since RLuc is unstable under various conditions. Therefore, an attempt was made to increase RLuc thermostability. In this study, 5 mutations reported previously [1] and one mutation obtained using site-directed mutagenesis were combined. As a result of this combination, the thermostability effect increased, with the mutant showing approximately 10 °C higher stability. Furthermore, the mutant simultaneously improved a tolerance for protease digestion, e.g. trypsin and proteinase K, and for organic solvent. Residual activity of the mutant after treatment with 10% 2-propanol, 10% DMF and 20% DMSO at 35 °C for 1 h was 29.4, 24.8 and 91.3%, respectively, whereas that of the wild type was 0.4, 0.1 and 24.3%, respectively.

Stabilization of luciferase from Renilla reniformis using random mutations.

We expressed luciferase (RLuc) from Renilla reniformis in Escherichia coli RLuc was purified using a Ni-NTA column and subsequently characterized. It was unstable in acidic solutions and at 30°C. To increase the stability of RLuc, the Rluc gene was randomly mutated using error-prone polymerase chain reaction. E. coli harboring the mutated gene was screened by detecting luminescence on a plate containing the substrate coelenterazine at 34°C. Three mutants, i.e. N264SS287P, N178D and F116LI137V, were obtained. The solubilities and specific activities of these mutants were higher than those of the wild type. Furthermore, the N264SS287P mutant maintained stability at a temperature approximately 5°C higher than that of the wild type, while denaturation of the F116LI137V mutant started at a temperature that was 5°C lower than the wild type, and ended at a temperature that was 7°C higher. We examined the obtained mutations using thermal shift assays and a computer program Coot in this study.

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody.

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 Å using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (-1.52 ± 0.05 kJ mol(-1)  K(-1) ) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (-1.02 ∼ 0.25 kJ mol(-1)  K(-1) ) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.

Nucleoside Diphosphate Kinase from Psychrophilic Pseudoalteromonas sp. AS-131 Isolated from Antarctic Ocean.

Nucleoside diphosphate kinase isolated from psychrophilic Pseudoalteromonas sp. AS-131 (ASNDK) was expressed in Escherichia coli and purified to homogeneity. Comparing to mesophilic NDK isolated from Pseudomonas aeruginosa, ASNDK exhibited highly elevated thermolability: E. coli expression at 37 °C as a denatured insoluble form, 30 °C lower optimum temperature of enzymatic activity, and greatly reduced heat stability with 38 °C lower Tm value, fourfold higher Km and reduced Kcat/Km by 0.4-fold upon reaction temperature increase from 20 to 37 °C. The subunit structure of ASNDK was suggested to be dimer, as in NDKs isolated from moderate halophiles.

Structure of a highly acidic ß-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs(+)-selective binding site.

Environmentally friendly absorbents are needed for Sr(2+) and Cs(+), as the removal of the radioactive Sr(2+) and Cs(+) that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs(+) or Sr(2+). The crystal structure of a halophilic ß-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Šin space group P31 using X-ray crystallography. Moreover, the locations of bound Sr(2+) and Cs(+) ions were identified by anomalous X-ray diffraction. The location of one Cs(+)-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na(+) (90 mM Na(+)/10 mM Cs(+)). From an activity assay using isothermal titration calorimetry, the bound Sr(2+) and Cs(+) ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs(+)-binding site provides important information that is useful for the design of artificial Cs(+)-binding sites that may be useful in the bioremediation of radioactive isotopes.

Structural characteristics of alkaline phosphatase from the moderately halophilic bacterium Halomonas sp. 593.

Alkaline phosphatase (AP) from the moderate halophilic bacterium Halomonas sp. 593 (HaAP) catalyzes the hydrolysis of phosphomonoesters over a wide salt-concentration range (1-4 M NaCl). In order to clarify the structural basis of its halophilic characteristics and its wide-range adaptation to salt concentration, the tertiary structure of HaAP was determined by X-ray crystallography to 2.1 Šresolution. The unit cell of HaAP contained one dimer unit corresponding to the biological unit. The monomer structure of HaAP contains a domain comprised of an 11-stranded ß-sheet core with 19 surrounding α-helices similar to those of APs from other species, and a unique `crown' domain containing an extended `arm' structure that participates in formation of a hydrophobic cluster at the entrance to the substrate-binding site. The HaAP structure also displays a unique distribution of negatively charged residues and hydrophobic residues in comparison to other known AP structures. AP from Vibrio sp. G15-21 (VAP; a slight halophile) has the highest similarity in sequence (70.0% identity) and structure (C(α) r.m.s.d. of 0.82 Šfor the monomer) to HaAP. The surface of the HaAP dimer is substantially more acidic than that of the VAP dimer (144 exposed Asp/Glu residues versus 114, respectively), and thus may enable the solubility of HaAP under high-salt conditions. Conversely, the monomer unit of HaAP formed a substantially larger hydrophobic interior comprising 329 C atoms from completely buried residues, whereas that of VAP comprised 264 C atoms, which may maintain the stability of HaAP under low-salt conditions. These characteristics of HaAP may be responsible for its unique functional adaptation permitting activity over a wide range of salt concentrations.

Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa.

∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75Å resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.

Reduction of salt-requirement of halophilic nucleoside diphosphate kinase by engineering S-S bond.

Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum, requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S-S bond. It showed increased thermal stability by about 10 °C in 0.2 M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1 M NaCl, while the wild type required 2 M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S-S bonds between two basic dimeric units in the hexameric HsNDK structure, indicating that assembly of the dimeric unit may be the rate-limiting step in low salt solution. Circular dichroism and native-PAGE analysis showed that heat-denatured HsNDK formed partially folded dimeric structure, upon refolding, in the absence of salt and the native-like secondary structure in the presence of salt above 0.1 M NaCl. However, it remained dimeric upon prolonged incubation at this salt concentration. In contrary, heat-denatured D148C mutant refolded into tetrameric folding intermediate in the absence of salt and native-like structure above 0.1 M salt. This native-like structure was then converted to the native hexamer with time.

A structural mechanism for dimeric to tetrameric oligomer conversion in Halomonas sp. nucleoside diphosphate kinase.

Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X-ray crystallography. The molecular weight of HaNDK is 33,660, and the X-ray crystal structure determination to 2.3 and 2.7 Å resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1-65.3 kDa, and the oligomerization state was investigated by X-ray crystallography to 2.3 and 2.5 Å resolution with space groups of P2(1) and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild-type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild-type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.

Promoter-associated noncoding RNA from the CCND1 promoter.

More than 90% of the human genome have been found to be transcribed and most of the transcripts are noncoding (nc) RNAs (Willingham et al., Science 309:1570-1573, 2005; ENCODE-consortium, Science 306:636-640, 2004; Carninci et al., Science 309:1559-1563, 2005; Bertone et al., Science 306:2242-2246, 2004). Studies on ncRNAs have been radically progressed mainly regarding microRNAs, piRNAs, siRNAs, and related small ncRNAs of which length are relatively short nucleotides (Fire et al., Nature 391:806-811, 1998; Filipowicz et al., Nat Rev Genet 9:102-114, 2008; Lau et al., Science 313:363-367, 2006; Brennecke et al., Science 322:1387-1392, 2008; Siomi and Siomi, Nature 457:396-404, 2009). These small RNAs play roles in regulation of translation and gene silencing while long ncRNAs with length more than 200 nucleotides have been emerging and turn out to be involved in regulation of transcription (Kapranov et al., Science 316:1484-1488, 2007; Ponting et al., Cell 136:629-641, 2009; Kurokawa et al., RNA Biol 6:233-236, 2009). Recently, we have identified novel, long ncRNAs bearing capability of repression of transcription (Wang et al., Nature 454:126-130, 2008).RNA-binding protein, translocated in liposarcoma (TLS), binds CREB-binding protein CBP/adenovirus p300 and inhibits their histone acetyltransferase (HAT) activities (Wang et al., Nature 454:126-130, 2008). The HAT inhibitory activity of TLS requires specific binding of RNA. The systematic evolution of ligands by exponential enrichment experiments with randomized sequences revealed that TLS specifically recognizes RNA oligonucleotides containing GGUG as a consensus sequence although the GGUG sequence is not an absolute requirement for the TLS binding (Lerga et al., J Biol Chem 276:6807-6816, 2001). TLS is specifically recruited to the CBP/p300-associated binding sites of the cyclin D1 gene (CCND1) and the cyclin E1 gene (CCNE1) promoters (Wang et al., Nature 454:126-130, 2008; Impey et al., Cell 119:1041-1054, 2004). Our extensive exploration for naturally occurring RNA molecule that binds TLS has indicated that long ncRNAs (promoter-associated ncRNAs: pncRNAs) transcribed from the CCND1 promoter bind TLS and inhibit the HAT activities on the sites to repress the transcription of the CCND1 gene (Wang et al., Nature 454:126-130, 2008). We have optimized RT-PCR, chromatin immunoprecipitation, RNA immunoprecipitation, and RNA gel-shift assay in order to detect these pncRNAs. The methods that we have developed successfully identified these low-abundant, long ncRNAs and provide the data showing that the CCND1 pncRNAs bind TLS and induce its HAT inhibitory activity to repress the transcription of CCND1 gene upon genotoxic stress.