Many protein-protein and protein-nucleic acid interactions have been experimentally characterized, whereas RNA-RNA interactions have generally only been predicted computationally. Here, we describe a high-throughput method to identify intramolecular and intermolecular RNA-RNA interactions experimentally by crosslinking, ligation, and sequencing of hybrids (CLASH). As validation, we identified 39 known target sites for box C/D modification-guide small nucleolar RNAs (snoRNAs) on the yeast pre-rRNA. Novel snoRNA-rRNA hybrids were recovered between snR4-5S and U14-25S. These are supported by native electrophoresis and consistent with previously unexplained data. The U3 snoRNA was found to be associated with sequences close to the 3′ side of the central pseudoknot in 18S rRNA, supporting a role in formation of this structure. Applying CLASH to the yeast U2 spliceosomal snRNA led to a revised predicted secondary structure, featuring alternative folding of the 3′ domain and long-range contacts between the 3′ and 5′ domains. CLASH should allow transcriptome-wide analyses of RNA-RNA interactions in many organisms.pre-rRNA | RNA structure | ribosome synthesis | UV cross-linking T he identification of RNA-RNA interactions is essential for detailed understanding of many biological processes. Almost all RNAs must be correctly folded to function, whereas basepairing between different RNA molecules underlies many pathways of RNA metabolism, including pre-mRNA splicing, ribosome synthesis, and the regulation of mRNA stability by microRNAs (miRNAs), among many others. Even for RNAs for which the final structure is known (e.g., rRNA), the folding pathway in precursors is generally unclear. RNA-RNA interactions were previously analyzed by X-ray crystallography, NMR, psoralen cross-linking, and genetics, but all these methods are labor-intensive and typically require prior knowledge of the interacting partners. Because of these technical difficulties, RNA base-pairing is more commonly inferred from a combination of bioinformatic and evolutionary analyses. However, computational methods are applicable only to evolutionarily conserved interactions and provide little information about the physiological context of the interaction.UV cross-linking methods have been developed to map protein interaction sites precisely on RNA molecules, including crosslinking and immunoprecipitation (CLIP) and cross-linking and analysis of cDNAs (CRAC) (1, 2). CRAC analyses have been performed on proteins (Nop1, Nop56, and Nop58) that are associated with all members of the box C/D class of small nucleolar RNAs (snoRNAs). Most box C/D snoRNAs base-pair with the rRNA to select sites of RNA 2′-O-methylation by the methyltransferase fibrillarin (Nop1). In contrast, the U3 snoRNA basepairs to multiple sites on the pre-rRNA. These interactions probably facilitate correct folding of the pre-rRNA and are required for pre-rRNA processing (3, reviewed in refs. 4, 5). PremRNA splicing requires five snRNAs that assemble the complex structure of the spliceosome, with...

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Accurate in vitro assessment of nanoparticle cytotoxicity requires a careful selection of the test systems. Due to high adsorption capacity and optical activity, engineered nanoparticles are highly potential in influencing classical cytotoxicity assays. Here, four common in vitro assays for oxidative stress, cell viability, cell death and inflammatory cytokine production (DCF, MTT, LDH and IL-8 ELISA) were assessed for validity using 24 well-characterized engineered nanoparticles. For all nanoparticles, the possible interference with the optical detection methods, the ability to convert the substrates, the influence on enzymatic activity and the potential to bind proinflammatory cytokines were analyzed in detail. Results varied considerably depending on the assay system used. All nanoparticles tested were found to interfere with the optical measurement at concentrations of 50 μg cm⁻² and above when DCF, MTT and LDH assays were performed. Except for Carbon Black, particle interference could be prevented by altering assay protocols and lowering particle concentrations to 10 μg cm⁻². Carbon Black was also found to oxidize H₂DCF-DA in a cell-free system, whereas only ZnO nanoparticles significantly decreased LDH activity. A dramatic loss of immunoreactive IL-8 was observed for only one of the three TiO₂ particle types tested. Our results demonstrate that engineered nanoparticles interfere with classic cytotoxicity assays in a highly concentration-, particle- and assay-specific manner. These findings strongly suggest that each in vitro test system has to be evaluated for each single nanoparticle type to accurately assess the nanoparticle toxicity.

Nanomaterials can display distinct biological effects compared with bulk materials of the same chemical composition. The physico-chemical characterization of nanomaterials and their interaction with biological media are essential for reliable studies and are reviewed here with a focus on widely used metal oxide and carbon nanomaterials. Available rat inhalation and cell culture studies compared to original results suggest that hazard potential is not determined by a single physico-chemical property but instead depends on a combination of material properties. Reactive oxygen species generation, fiber shape, size, solubility and crystalline phase are known indicators of nanomaterials biological impact. According to these properties the summarized hazard potential decreases in the order multi-walled carbon nanotubes >> CeO(2), ZnO > TiO(2) > functionalized SiO(2) > SiO(2), ZrO(2), carbon black. Enhanced understanding of biophysical properties and cellular effects results in improved testing strategies and enables the selection and production of safe materials.