Cytochrome c oxidase deficiency detection in human fibroblasts using scanning electrochemical microscopy.

Cytochrome c oxidase deficiency (COXD) is an inherited disorder characterized by the absence or mutation in the genes encoding for the cytochrome c oxidase protein (COX). COX deficiency results in severe muscle weakness, heart, liver, and kidney disorders, as well as brain damage in infants and adolescents, leading to death in many cases. With no cure for this disorder, finding an efficient, inexpensive, and early means of diagnosis is essential to minimize symptoms and long-term disabilities. Furthermore, muscle biopsy, the traditional detection method, is invasive, expensive, and time-consuming. This study demonstrates the applicability of scanning electrochemical microscopy to quantify COX activity in living human fibroblast cells. Taking advantage of the interaction between the redox mediator N, N, N', N'-tetramethyl-para-phenylene-diamine, and COX, the enzymatic activity was successfully quantified by monitoring current changes using a platinum microelectrode and determining the apparent heterogeneous rate constant k0 using numerical modeling. This study provides a foundation for developing a diagnostic method for detecting COXD in infants, which has the potential to increase treatment effectiveness and improve the quality of life of affected individuals.

Nuclear SRP9/SRP14 heterodimer transcriptionally regulates 7SL and BC200 RNA expression.

The SRP9/SRP14 heterodimer is a central component of signal recognition particle (SRP) RNA (7SL) processing and Alu retrotransposition. In this study, we sought to establish the role of nuclear SRP9/SRP14 in the transcriptional regulation of 7SL and BC200 RNA. 7SL and BC200 RNA steady-state levels, rate of decay, and transcriptional activity were evaluated under SRP9/SRP14 knockdown conditions. Immunofluorescent imaging, and subcellular fractionation of MCF-7 cells, revealed a distinct nuclear localization for SRP9/SRP14. The relationship between this localization and transcriptional activity at 7SL and BC200 genes was also examined. These findings demonstrate a novel nuclear function of SRP9/SRP14 establishing that this heterodimer transcriptionally regulates 7SL and BC200 RNA expression. We describe a model in which SRP9/SRP14 cotranscriptionally regulate 7SL and BC200 RNA expression. Our model is also a plausible pathway for regulating Alu RNA transcription and is consistent with the hypothesized roles of SRP9/SRP14 transporting 7SL RNA into the nucleolus for posttranscriptional processing, and trafficking of Alu RNA for retrotransposition.

Distinct domain organization and diversity of 2'-5'-oligoadenylate synthetases.

The 2'-5'-oligoadenylate synthetases (OAS) are important components of the innate immune system that recognize viral double-stranded RNA (dsRNA). Upon dsRNA binding, OAS generate 2'-5'-linked oligoadenylates (2-5A) that activate ribonuclease L (RNase L), halting viral replication. The OAS/RNase L pathway is thus an important antiviral pathway and viruses have devised strategies to circumvent OAS activation. OAS enzymes are divided into four classes according to size: small (OAS1), medium (OAS2), and large (OAS3) that consist of one, two, and three OAS domains, respectively, and the OAS-like protein (OASL) that consists of one OAS domain and tandem domains similar to ubiquitin. Early investigation of the OAS enzymes hinted at the recognition of dsRNA by OAS, but due to size differences amongst OAS family members combined with the lack of structural information on full-length OAS2 and OAS3, the regulation of OAS catalytic activity by dsRNA was not well understood. However, the recent biophysical studies of OAS have highlighted overall structure and domain organization. In this review, we present a detailed examination of the OAS literature and summarized the investigation on 2'-5'-oligoadenylate synthetases.

Photoactive monofunctional platinum(II) anticancer complexes of multidentate phenanthridine-containing ligands: photocytotoxicity and evidence for interaction with DNA.

Pt(II) complexes supported by chelating, multidentate ligands containing π-extended, planar phenanthridine (benzo[c]quinoline) donors (RLPtCl) exhibit a promising in vitro therapeutic index compared with phenanthriplatin, a leading preclinical anticancer complex containing a monodentate phenanthridine ligand. Here, we report evidence for non-specific interactions of CF3LPtCl with DNA through intercalation-mediated turn-on luminescence in O2-saturated aqueous buffer. Brief irradiation with visible light (490 nm) was also found to drastically increase the activity of CF3LPtCl, with photocytotoxicity increased up to 87% against a variety of human cancer cell lines. Mechanistic studies highlight significantly improved cellular uptake of CF3LPtCl compared with cisplatin, with localization in the nucleus and mitochondria triggering effective apoptosis. Photosensitization experiments with 1,3-diphenylisobenzofuran demonstrate that CF3LPtCl efficiently mediates the generation of singlet dioxygen (1O2), highlighting the potential of RLPtCl in photodynamic therapy.

The noncoding RNA BC200 associates with polysomes to positively regulate mRNA translation in tumor cells.

BC200 is a noncoding RNA elevated in a broad spectrum of tumor cells that is critical for cell viability, invasion, and migration. Overexpression studies have implicated BC200 and the rodent analog BC1 as negative regulators of translation in both cell-based and in vitro translation assays. Although these studies are consistent, they have not been confirmed in knockdown studies and direct evidence for this function is lacking. Herein, we have demonstrated that BC200 knockdown is correlated with a decrease in global translation rates. As this conflicts with the hypothesis that BC200 is a translational suppressor, we overexpressed BC200 by transfection of in vitro transcribed RNA and transient expression from transfected plasmids. In this context BC200 suppressed translation; however, an innate immune response confounded the data. To overcome this, breast cancer cells stably overexpressing BC200 and various control RNAs were developed by selection for genomic incorporation of a plasmid coexpressing BC200 and the neomycin resistance gene. Stable overexpression of BC200 was associated with elevated translation levels in pooled stable cell lines and isolated single-cell clones. Cross-linking sucrose density gradient centrifugation demonstrated an association of BC200 and its reported binding partners SRP9/14, CSDE1, DHX36, and PABPC1 with both ribosomal subunits and polysomal RNA, an association not previously observed owing to the labile nature of the interactions. In summary, these data present a novel understanding of BC200 function as well as optimized methodology that has far reaching implications in the study of noncoding RNAs, particularly within the context of translational regulatory mechanisms.

An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21.

DDX21 is a newly discovered RNA G-quadruplex (rG4) binding protein with no known biological rG4 targets. In this study we used label-free proteomic MS/MS to identify 26 proteins that are expressed at significantly different levels in cells expressing an rG4-binding deficient DDX21 (M4). MS data are available via ProteomeXchange with identifier PXD013501. From this list we validate MAGED2 as a protein that is regulated by DDX21 through rG4 in its 5'-UTR. MAGED2 protein levels, but not mRNA levels, are reduced by half in cells expressing DDX21 M4. MAGED2 has a repressive effect on TRAIL-R2 expression that is relieved under these conditions, resulting in elevated TRAIL-R2 mRNA and protein in MCF-7 cells, rendering them sensitive to TRAIL-mediated apoptosis. Our work identifies the role of DDX21 in regulation at the translational level through biologically relevant rG4 and shows that MAGED2 protein levels are regulated, at least in part, by the potential to form rG4 in their 5'-UTRs.

Predicting human RNA quadruplex helicases through comparative sequence approaches and helicase mRNA interactome analyses.

RNA quadruplexes are non-canonical nucleic acid structures involved in several human disease states and are regulated by a specific subset of RNA helicases. Given the difficulty in identifying RNA quadruplex helicases due to the multifunctionality of these enzymes, we sought to provide a comprehensive in silico analysis of features found in validated RNA quadruplex helicases to predict novel human RNA quadruplex helicases. Using the 64 human RNA helicases, we correlated their amino acid compositions with subsets of RNA quadruplex helicases categorized by varying levels of evidence of RNA quadruplex interaction. Utilizing phylogenetic and synonymous/non-synonymous substitution analyses, we identified an evolutionarily conserved pattern involving predicted intrinsic disorder and a previously identified motif. We analyzed available next-generation sequencing data to determine which RNA helicases directly interacted with predicted RNA quadruplex regions intracellularly and elucidated the relationship with miRNA binding sites adjacent to RNA quadruplexes. Finally, we performed a phylogenetic analysis of all 64 human RNA helicases to establish how RNA quadruplex detection and unwinding activity may be conserved among helicase subfamilies. This work furthers the understanding of commonalities between RNA quadruplex helicases and provides support for the future validation of several human RNA helicases.

Alu RNA and their roles in human disease states.

Alu RNA are implicated in the poor prognosis of several human disease states. These RNA are transcription products of primate specific transposable elements called Alu elements. These elements are extremely abundant, comprising over 10% of the human genome, and 100 to 1000 cytoplasmic copies of Alu RNA per cell. Alu RNA do not have a single universal functional role aside from selfish self-propagation. Despite this, Alu RNA have been found to operate in a diverse set of translational and transcriptional mechanisms. This review will focus on the current knowledge of Alu RNA involved in human disease states and known mechanisms of action. Examples of Alu RNA that are transcribed in a variety of contexts such as introns, mature mRNA, and non-coding transcripts will be discussed. Past and present challenges in studying Alu RNA, and the future directions of Alu RNA in basic and clinical research will also be examined.

Structural and Hydrodynamic Characterization of Dimeric Human Oligoadenylate Synthetase 2.

Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2'-5'-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2'-5'-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.

Impact of double-stranded RNA characteristics on the activation of human 2'-5'-oligoadenylate synthetase 2 (OAS2).

Human 2'-5' oligoadenylate synthetases (OAS) are a family of interferon-inducible proteins that, upon activation by double-stranded RNA, polymerize ATP into 2'-5' linked oligoadenylates. In this study, we probed the RNA cofactor specificity of the two smallest isozymes, OAS1 and OAS2. First, we developed a strategy for the expression and purification of recombinant human OAS2 from eukaryotic cells and quantified the activity of the enzyme relative to OAS1 in vitro. We then confirmed that both OAS2 domains, as opposed to only the domain containing the canonical catalytic aspartic acid triad, are required for enzymatic activity. Enzyme kinetics of both OAS1 and OAS2 in the presence of a variety of RNA binding partners enabled characterization of the maximum reaction velocity and apparent RNA-protein affinity of activating RNAs. While in this study OAS1 can be catalytically activated by dsRNA of any length greater than 19 bp, OAS2 showed a marked increase in activity with increasing dsRNA length with a minimum requirement of 35 bp. Interestingly, activation of OAS2 was also more efficient when the dsRNA contained 3'-overhangs, despite no significant impact on binding affinity. Highly structured viral RNAs that are established OAS1 activators were not able to activate OAS2 enzymatic activity based on the lack of extended stretches of dsRNA of greater than 35 bp. Together these results may highlight distinct subsets of biological RNAs to which different human OAS isozymes respond.

Comprehensive analysis of the BC200 ribonucleoprotein reveals a reciprocal regulatory function with CSDE1/UNR.

BC200 is a long non-coding RNA primarily expressed in brain but aberrantly expressed in various cancers. To gain a further understanding of the function of BC200, we performed proteomic analyses of the BC200 ribonucleoprotein (RNP) by transfection of 3' DIG-labelled BC200. Protein binding partners of the functionally related murine RNA BC1 as well as a scrambled BC200 RNA were also assessed in both human and mouse cell lines. Stringent validation of proteins identified by mass spectrometry confirmed 14 of 84 protein binding partners and excluded eight proteins that did not appreciably bind BC200 in reverse experiments. Gene ontology analyses revealed general roles in RNA metabolic processes, RNA processing and splicing. Protein/RNA interaction sites were mapped with a series of RNA truncations revealing three distinct modes of interaction involving either the 5' Alu-domain, 3' A-rich or 3' C-rich regions. Due to their high enrichment values in reverse experiments, CSDE1 and STRAP were further analyzed demonstrating a direct interaction between CSDE1 and BC200 and indirect binding of STRAP to BC200 via heterodimerization with CSDE1. Knock-down studies identified a reciprocal regulatory relationship between CSDE1 and BC200 and immunofluorescence analysis of BC200 knock-down cells demonstrated a dramatic reorganization of CSDE1 into distinct nuclear foci.

Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge.

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5'-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.

Human DDX21 binds and unwinds RNA guanine quadruplexes.

Guanine quadruplexes (G4s) are an important structure of nucleic acids (DNA and RNA) with roles in several cellular processes. RNA G4s require specialized unwinding enzymes, of which only two have been previously identified. We describe the results of a simple and specific mass spectrometry guided method used to screen HEK293T cell lysate for G4 binding proteins. From these results, we validated the RNA helicase protein DDX21. DDX21 is an established RNA helicase, but has not yet been validated as a G4 binding protein. Through biochemical techniques, we confirm that DDX21-quadruplex RNA interactions are direct and mediated via a site of interaction at the C-terminus of the protein. Furthermore, through monitoring changes in nuclease sensitivity we show that DDX21 can unwind RNA G4. Finally, as proof of principle, we demonstrate the ability of DDX21 to suppress the expression of a protein with G4s in the 3΄ UTR of its mRNA.

Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers.

The effective tracking and purification of biological RNAs and RNA protein complexes is currently challenging. One promising strategy to simultaneously address both of these problems is to develop high-affinity RNA aptamers against taggable small molecule fluorophores. RNA Mango is a 39-nucleotide, parallel-stranded G-quadruplex RNA aptamer motif that binds with nanomolar affinity to a set of thiazole orange (TO1) derivatives while simultaneously inducing a 103-fold increase in fluorescence. We find that RNA Mango has a large increase in its thermal stability upon the addition of its TO1-Biotin ligand. Consistent with this thermal stabilization, RNA Mango can effectively discriminate TO1-Biotin from a broad range of small molecule fluorophores. In contrast, RNA Spinach, which is known to have a substantially more rigid G-quadruplex structure, was found to bind to this set of fluorophores, often with higher affinity than to its native ligand, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), and did not exhibit thermal stabilization in the presence of the TO1-Biotin fluorophore. Our data suggest that RNA Mango is likely to use a concerted ligand-binding mechanism that allows it to simultaneously bind and recognize its TO1-Biotin ligand, whereas RNA Spinach appears to lack such a mechanism. The high binding affinity and fluorescent efficiency of RNA Mango provides a compelling alternative to RNA Spinach as an RNA reporter system and paves the way for the future development of small fluorophore RNA reporter systems.

Insights into the RNA quadruplex binding specificity of DDX21.

Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 directly interacts with TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2'OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2'OH of loop nucleotides.

Structural studies of RNA-protein complexes: A hybrid approach involving hydrodynamics, scattering, and computational methods.

The diverse functional cellular roles played by ribonucleic acids (RNA) have emphasized the need to develop rapid and accurate methodologies to elucidate the relationship between the structure and function of RNA. Structural biology tools such as X-ray crystallography and Nuclear Magnetic Resonance are highly useful methods to obtain atomic-level resolution models of macromolecules. However, both methods have sample, time, and technical limitations that prevent their application to a number of macromolecules of interest. An emerging alternative to high-resolution structural techniques is to employ a hybrid approach that combines low-resolution shape information about macromolecules and their complexes from experimental hydrodynamic (e.g. analytical ultracentrifugation) and solution scattering measurements (e.g., solution X-ray or neutron scattering), with computational modeling to obtain atomic-level models. While promising, scattering methods rely on aggregation-free, monodispersed preparations and therefore the careful development of a quality control pipeline is fundamental to an unbiased and reliable structural determination. This review article describes hydrodynamic techniques that are highly valuable for homogeneity studies, scattering techniques useful to study the low-resolution shape, and strategies for computational modeling to obtain high-resolution 3D structural models of RNAs, proteins, and RNA-protein complexes.

Nanoscale Assembly of High-Mobility Group AT-Hook 2 Protein with DNA Replication Fork.

High mobility group AT-hook 2 (HMGA2) protein is composed of three AT-hook domains. HMGA2 expresses at high levels in both embryonic stem cells and cancer cells, where it interacts with and stabilizes replication forks (RFs), resulting in elevated cell proliferation rates. In this study, we demonstrated that HMGA2 knockdown reduces cell proliferation. To understand the features required for interaction between HMGA2 and RFs, we studied the solution structure of HMGA2, free and in complex with RFs, using an integrated host of biophysical techniques. Circular dichroism and NMR experiments confirmed the disordered state of unbound HMGA2. Dynamic light scattering and sedimentation velocity experiments demonstrated that HMGA2 and RF are monodisperse in solution, and form an equimolar complex. Small-angle x-ray scattering studies revealed that HMGA2 binds in a side-by-side orientation to RF where 3 AT-hooks act as a clamp to wrap around a distorted RF. Thus, our data provide insights into how HMGA2 interacts with stalled RFs and the function of the process.

RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3'-Tail of the Long Non-coding RNA BC200 (BCYRN1).

RNA helicase associated with AU-rich element (RHAU) is an ATP-dependent RNA helicase that demonstrates high affinity for quadruplex structures in DNA and RNA. To elucidate the significance of these quadruplex-RHAU interactions, we have performed RNA co-immunoprecipitation screens to identify novel RNAs bound to RHAU and characterize their function. In the course of this study, we have identified the non-coding RNA BC200 (BCYRN1) as specifically enriched upon RHAU immunoprecipitation. Although BC200 does not adopt a quadruplex structure and does not bind the quadruplex-interacting motif of RHAU, it has direct affinity for RHAU in vitro. Specifically designed BC200 truncations and RNase footprinting assays demonstrate that RHAU binds to an adenosine-rich region near the 3'-end of the RNA. RHAU truncations support binding that is dependent upon a region within the C terminus and is specific to RHAU isoform 1. Tests performed to assess whether BC200 interferes with RHAU helicase activity have demonstrated the ability of BC200 to act as an acceptor of unwound quadruplexes via a cytosine-rich region near the 3'-end of the RNA. Furthermore, an interaction between BC200 and the quadruplex-containing telomerase RNA was confirmed by pull-down assays of the endogenous RNAs. This leads to the possibility that RHAU may direct BC200 to bind and exert regulatory functions at quadruplex-containing RNA or DNA sequences.

The long non-coding RNA BC200 (BCYRN1) is critical for cancer cell survival and proliferation.

BACKGROUND: BC200 is a long non-coding RNA expressed at high levels in the brain and elevated in a variety of tumour types. BC200 has a hypothesized role in translational regulation; however, to date the functional role of BC200 in both normal and diseased states remains poorly characterized. METHODS: Detailed BC200 expression analyses were performed in tumor cell lines, primary and non-tumorigenic cultured breast and lung cells, and a panel of normal human tissues by quantitative real-time PCR and confirmed by northern blot. Subcellular fractionation was performed to assess BC200 distribution and efficient knock-down of BC200 was established using both locked nucleic acid (LNA) GapmeRs and conventional siRNAs. Cell viability following BC200 knockdown and overexpression was assessed by MTT assay and induction of apoptosis was monitored by Annexin V/PI staining and flow cytometry. Cell cycle arrest and synchronization were performed using serum withdrawal as well as the specific inhibitors Lovastatin, Thymidine, RO3306 and Nocodazole. Synchronization was monitored by fluorescent analysis of cellular DNA content by flow cytometry RESULTS: BC200 expression was substantially upregulated in brain and elevated expression was also observed in testes, small intestine and ovary. Expression in cultured tumour cells was dramatically higher than corresponding normal tissue; however, expression in cultured primary cells was similar to that in immortalized and cancer cell lines. BC200 knockdown resulted in a dramatic loss of viability through growth arrest and induction of apoptosis that could be partially rescued by overexpression of wild-type BC200 but not an siRNA-resistant sequence mutant. A substantial decrease in BC200 expression was observed upon cell confluence or serum deprivation, as well as drug induced cell cycle arrest in G1 or G2 but not S- or M-phases. Upon release from cell cycle arrest, BC200 expression was recovered as cells entered S-phase, but did not follow a periodic expression pattern during synchronized progression through the cell cycle. This elevated expression was critical for the survival of proliferating cancerous and non-cancerous cells, but is dispensable upon senescence or cell cycle arrest. CONCLUSIONS: BC200 expression is elevated in proliferating cultured cells regardless of origin. In primary cells, expression is dramatically reduced upon cell cycle arrest by confluence, serum deprivation or chemical inhibition. The lethality of BC200 knockdown is restricted to actively proliferating cells, making it a promising therapeutic target for a broad spectrum of cancers.

Impact of G-quadruplex loop conformation in the PITX1 mRNA on protein and small molecule interaction.

Intramolecular G-quadruplexes (G4s) are G-rich nucleic acid structures that fold back on themselves via interrupting loops to create stacked planar G-tetrads, in which four guanine bases associate via Hoogsteen hydrogen bonding. The G4 structure is further stabilized by monovalent cations centered between the stacked tetrads. The G-tetrad face on the top and bottom planes of G4s are often the site of interaction with proteins and small molecules. To investigate the potential impact of interrupting loops on both G4 structure and interaction with proteins/small molecules, we characterized a specific G4 from the 3'-UTR of PITX1 mRNA that contains loops of 6 nucleotides using biophysical approaches. We then introduced mutations to specific loops to determine the impact on G4 structure and the ability to interact with both proteins and a G4-specific ligand. Our results suggest that mutation of a specific loop both affects the global G4 structure and impacts the ability to interact with a G4 binding protein and small molecule ligand.