HeLa Cell Nuclear Extracts Research Articles

Abstract LB273: Pharmacological synthetic lethality by co-inhibition of PARP and HDAC enzymes

Abstract Introduction: Inhibition of poly(ADP-ribose) polymerase (PARP) induces synthetic lethality in cells exhibiting defects in homologous repair (HR) pathways, such as BRCA1 and BRCA2-mutated breast or ovarian cancers, and 4 different PARP inhibitors (PARPi) are currently approved by the FDA. While HR-deficient cancers usually respond well to single agent PARPi, HR-proficient cancers are generally less sensitive to the treatment. Acquired resistance to PARPi due to activating mutations in BRCA genes are also not uncommon. In this work, we have examined strategies for enhancing PARPi activity in a HR-proficient context and identified histone deacetylases (HDACs) as targets for pharmacological synthetic lethality in the context of PARPi. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50 were determined using CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by immunoblotting and immunofluorescence using gamma-H2AX antibodies, and comet assays for confirmation of double-strand breaks. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module. In vivo metastasis formations were examined using the pulmonary metastasis assay (PuMA). Results and discussion: Combinations of PARP and HDAC inhibitors in HR-proficient tumor cells demonstrated that HDACi could enhance the efficacy of PARPi in vitro. This was associated with increased acute and sustained DNA-damage distinct cell cycle profiles showing activity in both S- and G2/M-phases. Importantly, dual single-molecule PARP-HDAC inhibitors showed potent inhibition of PARP1 and PARP2 activity and PAR synthesis, comparable to olaparib, as well as inhibition of HDAC with IC50 values in the low µM range. Dual PARP-HDAC inhibition decreased survival of HR-proficient cells in both 2D cultures and 3D spheroid assays with lower EC50 values than olaparib or vorinostat alone. Importantly, lung metastasis formation was significantly impaired by dual PARP-HDAC inhibition. Conclusion: Combination of PARP inhibition and HDAC inhibition showed increased efficacy compared to single agent PARP inhibition in HR-proficient cells. Development of bifunctional inhibitors may provide a novel therapeutic opportunity for tumors with limited response rates to single agent PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Fariba Ghaidi, Mona Marzban, Hans Adomat, Xiaoqi Chen, John Langlands, Dennis Brown, Jeffrey Bacha, Colin Collins, Poul H. Sorensen, Wang Shen, Mads Daugaard. Pharmacological synthetic lethality by co-inhibition of PARP and HDAC enzymes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB273.

Pyridine Derivative of Succinic Acid Hydroxylamide Enhances the Cytotoxic Effect of Cisplatin and Actinomycin D.

We studied the possibility of inhibition of histone deacetylases (HDAC) in the nuclear extract of HeLa cells by N1-hydroxy-N4-(pyridin-4-yl)succinamide (compound 1). Compound 1 inhibits HDAC and showed low toxicity for A-172, HepG2, HeLa, MCF-7, and Vero cells. HeLa cells were most sensitive to the compound. Increasing the interval between administration of compound 1 and the chemotherapeutic agent to 8 h led to an increase in the cytotoxic effect of cisplatin (actinomycin D) on HeLa cells. The combination of compound 1 with cisplatin (actinomycin D) reduced the cytotoxic effect of these drugs for non-tumor Vero cells.

Abstract 6194: A bifunctional inhibitor of PARP and HDAC enzymes with activity in Ewing sarcoma 3D spheroid and metastasis models

Abstract Introduction: Poly(ADP-ribose) polymerase (PARP) plays a major role in DNA repair and PARP inhibitors (PARPi) have shown promise in pre-clinical studies for the treatment of Ewing sarcoma (ES). While a clinical trial using olaparib as a single agent failed to show significant response against ES, combination therapies with PARPi have emerged as an area of interest. Deacetylation of histones, controlled by histone deacetylases (HDACs) is a key regulatory event in DNA repair and inhibition of HDACs has been shown to reduce ES tumor growth in vitro and in vivo. PARP inhibition combined with HDAC inhibition has demonstrated enhanced efficacy in pre-clinical studies in various tumor indications, and a clinical trial of olaparib and vorinostat combination therapy against metastatic breast cancer is currently ongoing. However, combination therapies can be limited in clinical utility due to overlapping toxicities and different pharmacokinetic profiles. Here, we report the efficacy of a novel bifunctional small-molecule compound, kt-3283, designed to have both PARP and HDAC inhibitory activities. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte® S3 system and CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by western blot, immunofluorescence, and comet assay. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module and inhibition of metastases was assessed in a PUMA ES mouse model. Results and discussion: Kt-3283 showed potent inhibition of PARP1/2 activity and PAR synthesis with IC50 values comparable to olaparib. Kt-3283 also showed inhibition of HDACs with an IC50 value in the low µM range. Cell survival EC50 values for the compound were also superior to those of olaparib and vorinostat in ES cell lines. Cell cycle and DNA damage analyses indicated S/G2/M cell cycle arrest and strong DNA damage upon treatment with kt-3283 at lower concentration range compared to olaparib and vorinostat. This compound also exhibited potent inhibition of 3D spheroid growth of ES cells with low µM EC50 values, and inhibited metastatic growth in a PUMA mouse model. Conclusion: Kt-3283 shows potent inhibition of PARP1/2 and HDAC activities. It induces S and G2/M cell cycle arrest and DNA damage, and inhibits 3D spheroid growth and metastatic potential of ES cells. Further investigation of this bifunctional single-molecule inhibitor may offer a novel treatment opportunity for ES and other solid tumors with limited responses to PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Jay Joshi, Fariba Ghaidi, Michael M. Lizardo, Taras Shyp, John Langlands, Dennis Brown, Jeffrey Bacha, Poul Sorensen, Wang Shen, Mads Daugaard. A bifunctional inhibitor of PARP and HDAC enzymes with activity in Ewing sarcoma 3D spheroid and metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6194.

109 (PB099) - A novel dual PARP-HDAC inhibitor for treatment of Ewing sarcoma

Background: Inhibition of poly-adenosine diphosphate-ribose polymerase (PARP) is an effective therapy against cancers with DNA damage repair (DDR) deficiencies, such as BRCA1 and BRCA2 defects. In preclinical studies, PARP inhibitors demonstrated potential therapeutic value in Ewing sarcoma (ES), but clinical trials with olaparib failed to show significant clinical benefit. A key regulatory event in DNA damage repair is acetylation and deacetylation of histones, controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increased expression of HDACs correlate with more malignant phenotypes in sarcomas and inhibition of HDAC in pre-clinical models of ES inhibits tumor growth. HDAC inhibition combined with PARP inhibition has promising results in vitro, however clinically, combination therapies often require complicated sequential dosing protocols due to different pharmacokinetic profiles and overlapping toxicities, that severely limits clinical utility. Here, we report the efficacy of a novel bifunctional small-molecule compound designed to have both PARP and HDAC inhibition activity. Methods: PARP1 and PARP2 activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit, the BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and by PARylation assays. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50 s were determined using live cell imaging with an Incucyte S3 system and the CellTiter Glo viability assay. DNA damage was detected by western blot, immuno-fluorescence, and comet assays. Results: A representative compound from the kt-3000 series showed potent inhibition of PARP1 and PARP2 with IC50 values in the low nM range, comparable to FDA-approved PARP inhibitors. The compound also showed inhibition of HDAC enzymes with IC50 values in the low μM range, slightly lower than the FDA-approved HDAC inhibitor, vorinostat. Cell survival EC50 values were superior to olaparib in ES cell lines in vitro. Treatment with the kt-3000 compound also resulted in increased DNA damage and S and G2/M cell cycle arrest as compared to olaparib. Conclusion: Our kt-3000 compound shows potent inhibition of PARP1, PARP2, and HDAC, as well as induction of DNA damage and cell cycle arrest. Further development of these bifunctional single molecule inhibitors may offer a novel treatment opportunity for Ewing sarcoma and other solid tumors with limited responses to PARPi monotherapies. Conflict of interest: Board of Directors: J. Bacha and D. Brown are directors of Rakovina Therapeutics. Corporate-sponsored Research: S. Truong, B. Zhai, F. Ghaidi, L. Ramos, J. Joshi are employees of Rakovina Therapeutics. J. Langlands, M. Daugaard and W. Shen are consultants to Rakovina Therapeutics.

Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy3. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers4. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5′-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.

Galectin-3-U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts.

Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.

Identifying Cytoplasmic and Nuclear C‐Terminal to LisH (CTLH) Complex Interactors Using High‐Throughput Proteomics

The C-terminal to LisH (CTLH) complex is a tumour-suppressive E3 Really Interesting New Gene (RING) ubiquitin ligase that regulates intracellular signaling pathways and maintains cellular homeostasis. It is comprised of 8 individual proteins that have been shown to mediate a variety of intracellular protein-protein interactions; of these proteins, Required for Meiotic Division 5 Homolog A (RMND5A) and Macrophage Erythroblast Attacher (MAEA) contain the RING domains required for the CTLH complex to mediate ubiquitin transfer to specific protein substrates. Although its E3 ligase activity has been recently characterized, the endogenous subcellular localization of the complex and compartment-specific protein interaction network – which encompasses many unidentified E3 ligase substrates – has not been identified. In this study, we hypothesized that the CTLH complex is present in the cytoplasm and nucleus, and that the CTLH complex mediates compartment-specific protein-protein interactions with cytoplasmic and nuclear proteins. To test this hypothesis, we used a chemical-based subcellular fractionation method combined with low- and high-throughput proteomics. We first optimized the chemical fractionation method and used Western blot to detect each endogenous CTLH complex member in HeLa cell cytoplasmic and nuclear protein extracts. We then identified compartment-specific CTLH complex interactors using Affinity Purification Coupled to Mass Spectrometry (AP-MS) using the main CTLH complex scaffold protein – Ran Binding Protein M (RanBPM) – as bait in cytoplasmic and nuclear HeLa protein extracts. Following Liquid Chromatography Electrospray Ionizing Tandem Mass Spectrometry (LC-ESI-MS/MS) and subsequent in silico filtering using the MiST algorithm, we identified 27 cytoplasmic, 155 nuclear, and 31 nucleocytoplasmic high-confidence (MiST Score ≥ 0.75) RanBPM interactors. STRING enrichment analysis of the high-confidence interactors revealed that RanBPM and its associated CTLH complex may be involved in a variety of conserved Gene Ontology (GO) processes including Epigenetic Regulation of Gene Expression and Chromatin Assembly. As such, we validated a number of newly-identified compartment-specific RanBPM interactors associated with the enriched GO terms by co-immunoprecipitation – using RanBPM as bait – and resulting Western blot. Furthermore, we focussed on an interesting novel putative interactor that is present in the two aforementioned GO processes – histone variant MacroH2A1 – and further validated its interaction with the CTLH complex in vivo using Proximity Ligation Assay (PLA), suggesting that this interaction may be physiologically relevant. Overall, by using this workflow, we identified and validated a number of novel compartment-specific potential CTLH complex interactors in vitro, further validated a promising novel interaction between the CTLH complex and MacroH2A1 in vivo, and successfully implicated the CTLH complex in a number of previously unexplored cellular mechanisms.

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads.

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

Sulfonated calix[4]arene functionalized SiO2@TiO2 for recognition of lysine methylation

Lysine methylations are common protein post-translational modifications (PTMs), that play significant roles in regulating gene activities. Studies of their functions and connections with diseases have important values. However, due to the small variations from their native structures and very low component proportions, it is very difficult to extract methylated peptides from biological mixtures. In this research, a new material that utilizes sulfonated calix[4]arene (SC4A) as the recognition unit and silica coated with TiO2 as carrier, denoted as SiO2@TiO2@SC4A, was synthesized. The equilibrium binding experiments demonstrated that SiO2@TiO2@SC4A can identify lysine and arginine methylation and peptides with these methylated residues. The maximum isotherm binding capacities are 70.0, 55.9, 31.4 and 24.8 μmol g−1 for Lys(Me3), Lys(Me)2, Lys(Me) and Lys, respectively. It demonstrated that the higher the degree of methylation, the stronger the interactions.In addition, the analyses of high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) demonstrated that peptides with methylated lysine or arginine can be selectively extracted from spiked histone trypsin digestion. The recoveries for the spiked GGAK(Me)R, GGAKR(Me)2 and GGAK(Me)3R are 83%, 78%, and 84% respectively. The experiments from the nuclear extracts of HeLa cells also illustrated that SiO2@TiO2@SC4A holds a potential in the enrichment and identification of lysine methylations.

Potential Inhibitor Against Phase Separation, 1,6-hexanediol Specifically Binds to Beta Actin in Nuclear Extract of Human Cell Line

RNA-binding protein (RBP) TLS/FUS plays a major role in induction of phase separation/phase transition and aggregation in relation to familial amyotrophic lateral sclerosis (ALS). Recently, organelles without lipid-bilayer membrane including stress granule, Cajal body, and nucleolus are found to be formed by the phase separation. The phase separation is an event that solutions with two solvents separate into two distinctive phases. The phase separation is prone to have solid phase and forms harmful precipitation or aggregation against living cells, indicating that the phase separation has both benefit and risk on cellular programs. Thus, it is essential for utilization of the phase separation in divergent cellular programs to control or inhibit the undesirable precipitation in living cells. Here, we analyze an inhibitory mechanism of the phase separation and precipitation. Inhibition of the phase separation is one of a critical regulatory step to prevent dysregulation of the phase separation and resulting deleterious precipitations. An inhibitory agent against the phase separation, amphiphilic alcohol, 1,6-hexanediol (1,6-HD) has been reported to examine function of phase separations. Thus, affinity chromatography of biotinylated 1,6-HD is employed to identify an initial event induced by 1,6-HD. Upon successful synthesis of biotinylated compounds of 1,6-HD (bio-1,6-HD), the affinity chromatography with bio-1,6-HD has been established at this study. The bio-1,6-HD captured protein bands on SDS-PAGE gel from HeLa cell nuclear extracts. The bands were analyzed with mass spectrometric analyses, showing that the proteins should be cytoskeleton related proteins. Further analysis using specific antibodies revealed one of the bands as human beta actin. It has been shown that beta actin is involved in divergent cellular activities including the phase separation and also neuronal functions like long-term potentiation. Therefore, beta actin might initiate the 1,6-HD-induced inhibition of the precipitation, although more experiments should be required to test whether it actually works in living cells.

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-Way System for RNAP II Transcription/Splicing/microRNA Processing.

In the genome, primary microRNAs (pri-miRNAs) are encoded either as independent transcriptional units with their own promoters (intergenic miRNAs) or within the introns of other genes (intronic miRNAs). Here, we report two methods, one that we established for coupled RNAP II transcription and pri-miRNA processing and the other that is a three-way system for RNAP II transcription, pri-miRNA processing, and pre-mRNA splicing. In these systems, CMV-DNA constructs encoding the processing substrates are incubated in HeLa cell nuclear extracts in the presence of 32P-UTP to generate the nascent RNAP II transcripts, which are processed efficiently by the endogenous RNA processing machineries in nuclear extracts.

Affinity Profiles Categorize RNA-Binding Proteins into Distinctive Groups

Recently, we have established a novel assay to detect interaction of RNA-binding proteins (RBPs) with RNA, using biotinylated RNA oligos to capture RBPs with Western blot of specific antibodies against RBPs. The assay detects RNA binding more confidently than the traditional gel shift assay. Starting with completely randomized RNA oligos from 5mer through 12mer length, their binding was examined with HeLa cell nuclear extract (NE). Coomassie brilliant blue-based (CBB) staining did not detect any strong signal. Western blot analysis of typical six RBP antibodies showed four RBPs bound with the random RNA oligos. hnRNPUL2 bound to all from 5mer to 12mer of RNA oligos, while no TLS and hnRNPH1 signal was detected in the random RNA oligo samples. Next, base specificity was examined using sets of oligos of RNA fixed at G, A, U, and C (GAUC RNA oliogs). The RNA oligos fixed at “G” (G RNA oligos) have the most prominent protein bands. A, U, and C of RNA oligos were shown to bind less numbers of protein. Western blot indicated that hnRNPUL2 and hnRNPU bind all four oligos of GAUC at the 10mer length. Contrarily, TLS and hnRNPH1 have no binding with these oligos of GAUC. Then, poly G, A, U and C of RNA at the length of 100mer were tested to see binding profile of RBPs. The CBB staining of the fractions bound with these four polymers of RNA showed that more bands were bound than GAUC RNA oligos. hnRNPU bound well to poly G, A, and, U, but slightly less to poly C. Intriguingly, TLS and hnRNPH1 have binding only to poly G, and also to their common specific sites consisting of GGUG motifs. These data demonstrate that RNA binding is regulated with three factors, length, base composition, and sequence. Furthermore, hnRNPU and hnRNPUL2 have low specificity binding to RNAs, while TLS and hnRNPH1 exert high specific binding. These different propensities in bindings of RBPs are supposed to support specific biological roles in living cells.

Biotin-Lys-His Blocks Aggregation of RNA-binding Protein TLS, a Cause of Amyotrophic Lateral Sclerosis

RNA-binding protein TLS/FUS is a causative gene for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TLS mutations induce the propensity of TLS to form aggregates in motor neurons causing neuronal degenerative lesions to their necrosis. TLS is prone to be precipitated in high concentration around 10 mg/ml, while mutated TLS is suspected to be precipitated even lower or physiological concentration in the motor neurons. An unidentified agent from infections would cause formation of the precipitation of TLS through their surface antigens. The precipitation driven with agents might be attribute to a small compound appeared on the surface. Biotinylated isoxazole (BISOX) has been reported to be precipitated with divergent RNA-binding proteins including TLS in nuclear extracts of cultured mammalian cells. We have published a molecular model for crystal formation of BISOX with TLS providing a plat form for searching novel regulators to precipitate TLS in neuronal disorders. Because BISOX is an artificial compound, we have further explored to obtain naturally occurring agents to induce the TLS precipitation and generated a conceivable biological compound, biotin-Lys-His (BLH). We have examined the precipitation of BLH with HeLa cell nuclear extract, but did not detect any TLS signal. Then, we add BLH to reaction of BISOX with TLS, and serendipitously observed a robust inhibitory effect of BLH on the formation of crystal of BISOX with TLS. We employed in silico analysis to show how BLH blocks the crystal formation of BISOX with TLS. The computational analysis of the events presented a model that BLH should be incorporated into the crystal formation of BISOX but some steric hindrance placed by BLH blocks growing of the crystal of BISOX and TLS. These results provide the potentiality that BLH should block the aggregate formation of TLS in ALS, leading to a seed for drug discovery against ALS, although it needs future endeavor to find more compounds to have effect on the TLS aggregation.

When transcription goes on Holliday: Double Holliday junctions block RNA polymerase II transcription in vitro

Non-canonical DNA structures can obstruct transcription. This transcription blockage could have various biological consequences, including genomic instability and gratuitous transcription-coupled repair. Among potential structures causing transcription blockage are Holliday junctions (HJs), which can be generated as intermediates in homologous recombination or during processing of stalled replication forks. Of particular interest is the double Holliday junction (DHJ), which contains two HJs. Topological considerations impose the constraint that the total number of helical turns in the DNA duplexes between the junctions cannot be altered as long as the flanking DNA duplexes are intact. Thus, the DHJ structure should strongly resist transient unwinding during transcription; consequently, it is predicted to cause significantly stronger blockage than single HJ structures. The patterns of transcription blockage obtained for RNA polymerase II transcription in HeLa cell nuclear extracts were in accordance with this prediction. However, we did not detect transcription blockage with purified T7 phage RNA polymerase; we discuss a possible explanation for this difference. In general, our findings implicate naturally occurring Holliday junctions in transcription arrest.

Optimizing In Vitro Pre-mRNA 3' Cleavage Efficiency: Reconstitution from Anion-Exchange Separated HeLa Cleavage Factors and from Adherent HeLa Cell Nuclear Extract.

Eukaryotic RNA processing steps during mRNA maturation present the cell with opportunities for gene expression regulation. One such step is the pre-mRNA 3' cleavage reaction, which defines the downstream end of the 3' untranslated region and, in nearly all mRNA, prepares the message for addition of the poly(A) tail. The in vitro reconstitution of 3' cleavage provides an experimental means to investigate the roles of the various multi-subunit cleavage factors. Anion-exchange chromatography is the simplest procedure for separating the core mammalian cleavage factors. Here we describe a method for optimizing the in vitro reconstitution of 3' cleavage activity from the DEAE-sepharose separated HeLa cleavage factors and show how to ensure, or avoid, dependence on creatine phosphate. Important reaction components needed for optimal processing are discussed. We also provide an optimized procedure for preparing small-scale HeLa nuclear extracts from adherent cells for use in 3' cleavage in vitro.

Colocalization recognition-activated cascade signal amplification strategy for ultrasensitive detection of transcription factors

Transcription factors (TFs) bind to specific double-stranded DNA (dsDNA) sequences in the regulatory regions of genes to regulate the process of gene transcription. Their expression levels sensitively reflect cell developmental situation and disease state. TFs have become potential diagnostic markers and therapeutic targets of cancers and some other diseases. Hence, high sensitive detection of TFs is of vital importance for early diagnosis of diseases and drugs development. The traditional exonucleases-assisted signal amplification methods suffered from the false positives caused by incomplete digestion of excess recognition probes. Herein, based on a new recognition way-colocalization recognition (CR)-activated dual signal amplification, an ultrasensitive fluorescent detection strategy for TFs was developed. TFs-induced the colocalization of three split recognition components resulted in noticeable increases of local effective concentrations and hybridization of three split components, which activated the subsequent cascade signal amplification including strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). This strategy eliminated the false positive influence and achieved ultra-high sensitivity towards the purified NF-κB p50 with detection limit of 2.0×10−13M. Moreover, NF-κB p50 can be detected in as low as 0.21ngμL−1 HeLa cell nuclear extracts. In addition, this proposed strategy could be used for the screening of NF-κB p50 activity inhibitors and potential anti-NF-κB p50 drugs. Finally, our proposed strategy offered a potential method for reliable detection of TFs in medical diagnosis and treatment research of cancers and other related diseases.

Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone

Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques―photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis—are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins.

A 10S galectin-3-U1 snRNP complex assembles into active spliceosomes.

In previous studies, we reported that fractionation of HeLa cell nuclear extracts on glycerol gradients revealed an endogenous ∼10S particle that contained galectin-3 and U1 snRNP and this particle was sufficient to load the galectin polypeptide onto a pre-mRNA substrate. We now document that this interaction between the galectin-3–U1 snRNP particle and the pre-mRNA results in a productive spliceosomal complex, leading to intermediates and products of the splicing reaction. Nuclear extracts were depleted of U1 snRNP with a concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3–U1 snRNP particle, isolated by immunoprecipitation of the 10S region (fractions 3–5) of the glycerol gradient with anti-galectin-3 antibodies. In contrast, parallel anti-galectin-3 immunoprecipitation of free galectin-3 molecules not in a complex with U1 snRNP (fraction 1 of the same gradient), failed to restore splicing activity. These results indicate that the galectin-3–U1 snRNP-pre-mRNA ternary complex is a functional E complex and that U1 snRNP is required to assemble galectin-3 onto an active spliceosome.

A nucleolar role for the Fanconi anemia pathway protein, FANCI, in rDNA transcription

Fanconi anemia (FA) is a disease of DNA repair because proteins mutated in patients with FA are impaired in removing interstrand DNA crosslinks. We propose a new nucleolar role for the FA protein, FANCI, in ribosome biogenesis based on extensive preliminary data. Ribosome biogenesis is the process of making ribosomes that initiates in the cell nucleolus with the transcription and processing of the pre‐ribosomal RNA (rRNA) followed by the assembly of mature ribosomes. Immunoaffinity purification of FANCI from HeLa cell nuclear extract followed by mass spectrometry revealed co‐purification with nucleolar proteins required for ribosome biogenesis, including PES1. Using subcellular fractionation, we have found that FANCI is indeed enriched in the nucleolus. In particular, the deubiquitinated form of FANCI is by far the most predominant form of nucleolar FANCI, even under conditions of DNA damage by mitomycin C, which stimulate monoubiquitination of FANCI. Immunoprecipitation revealed that deubiquitinated FANCI, but not its binding partner in DNA repair, FANCD2, is co‐immunoprecipitated by the nucleolar protein, PES1. To probe a possible nucleolar function for FANCI, we depleted FANCI in human cells and observed reduced transcription by RNA polymerase I (RNAPI). In addition, FANCI is co‐immunoprecipitated by the large subunit of RNAPI, indicating a physical and functional role for FANCI in efficient pre‐rRNA transcription. Finally, our results demonstrate novel regulatory mechanisms by deubiquitinases on the ubiquitination state of FANCI in the nucleolus. While our model does not contest the well‐established underlying DNA repair defect observed in cells from FA patients, it does suggest a dual pathophysiology for FA that includes defects both in DNA repair and in ribosome biogenesis.Support or Funding InformationThis work is funded by the NIGMS and NCI.