RNA in situ conformation sequencing reveals novel long-range RNA structures with impact on splicing.

Over recent years, long-range RNA structure has emerged as a factor that is fundamental to alternative splicing regulation. An increasing number of human disorders are now being associated with splicing defects; hence it is essential to develop methods that assess long-range RNA structure experimentally. RNA in situ conformation sequencing (RIC-seq) is a method that recapitulates RNA structure within physiological RNA-protein complexes. In this work, we juxtapose pairs of conserved complementary regions (PCCRs) that were predicted in silico with the results of RIC-seq experiments conducted in seven human cell lines. We show statistically that RIC-seq support of PCCRs correlates with their properties, such as equilibrium free energy, presence of compensatory substitutions, and occurrence of A-to-I RNA editing sites and forked eCLIP peaks. Exons enclosed in PCCRs that are supported by RIC-seq tend to have weaker splice sites and lower inclusion rates, which is indicative of post-transcriptional splicing regulation mediated by RNA structure. Based on these findings, we prioritize PCCRs according to their RIC-seq support and show, using antisense nucleotides and minigene mutagenesis, that PCCRs in two disease-associated human genes, PHF20L1 and CASK, and also PCCRs in their murine orthologs, impact alternative splicing. In sum, we demonstrate how RIC-seq experiments can be used to discover functional long-range RNA structures, and particularly those that regulate alternative splicing.

Multiple competing RNA structures dynamically control alternative splicing in the human ATE1 gene.

The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.

Cyanine mitochondrial dye with slightly selective cytotoxicity against A549 cancerous cells.

Delocalized lipophilic cations (DLCs) are known as mitochondria-addressed molecules. Mitochondria targeting may provide opportunities for tumor detection. DLCs may have antioxidant or anticancer properties. In this study, we focused on the toxicity and localization of 2-[(E)-2-(5-fluoro-2-methyl-1H-indol-3-yl)ethenyl]-1,6-dimethylpyridin-1-ium iodide (62E2), which has recently been found as a novel cytotoxic fluorescent compound. The excitation maximum of 62E2 is 452 ± 10 nm and its emission maximum is 579 ± 10 nm. It is accumulated in the cells and stains mitochondria in nanomolar concentrations. 62E2 is cytotoxic and mitotoxic in low micromolar concentrations, and it demonstrates some selectivity of cytotoxicity against A549 cancer cells. The closest analog of 62E2 is F16, which is the fluorescent mitotoxic agent that has been described earlier as a potential anticancer agent. We hope that 62E2 described here is useful in expanding the diversity of cyanine fluorescent mitochondrial dyes and the analysis of their structure-activity relationships.

Dispirooxindoles Based on 2-Selenoxo-Imidazolidin-4-Ones: Synthesis, Cytotoxicity and ROS Generation Ability.

A regio- and diastereoselective synthesis of two types of dispiro derivatives of 2-selenoxoimidazolidin-4-ones, differing in the position of the nitrogen atom in the central pyrrolidine ring of the spiro-fused system-namely, 2-selenoxodispiro[imidazolidine-4,3'-pyrrolidine-2',3″-indoline]-2″,5-diones (5a-h) and 2-senenoxodispiro[imidazolidine-4,3'-pyrrolidine-4',3″-indoline]-2″,5-diones (6a-m)-were developed based on a 1,3-dipolar cycloaddition of azomethine ylides generated from isatin and sarcosine or formaldehyde and sarcosine to 5-arylidene or 5-indolidene-2-selenoxo-tetrahydro-4H-imidazole-4-ones. Selenium-containing dispiro indolinones generally exhibit cytotoxic activity near to the activity of the corresponding oxygen and sulfur-containing derivatives. Compounds 5b, 5c, and 5e demonstrated considerable in vitro cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) test (concentration of compounds that caused 50% death of cells (CC50) 7.6-8.7 µM) against the A549 cancer cell line with the VA13/A549 selectivity index 5.2-6.9; some compounds (5 and 6) increased the level of intracellular reactive oxygen species (ROS) in the experiment on A549 and PC3 cells using platinized carbon nanoelectrode. The tests for p53 activation for compounds 5 and 6 on the transcriptional reporter suggest that the investigated compounds can only have an indirect p53-dependent mechanism of action. For the compounds 5b, 6b, and 6l, the ROS generation may be one of the significant mechanisms of their cytotoxic action.

Synthesis and biological activity of 5-aryliden-2-thiohydantoin S-aryl derivatives.

Three new and complementary approaches to S-arylation of 2-thiohydantoins have been developed: copper-catalyzed cross coupling with either arylboronic acids or aryl iodides under mild conditions, or direct nucleophilic substitution in activated aryl halides. For 38 diverse compounds, reaction yields for all three methods have been determined. Selected by molecular docking, they have been tested on androgen receptor activation, and p53-Mdm2 regulation, and A549, MCF7, VA13, HEK293T, PC3, LnCAP cell lines for cytotoxicity, Two of them turned out to be promising as androgen receptor activators (likely by allosteric regulation), and another one is shown to activate the p53 cascade. It is hoped that 2-thiohydantoin S-arylidenes are worth further studies as biologically active compounds.

Conserved long-range base pairings are associated with pre-mRNA processing of human genes.

The ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. Current knowledge on functional RNA structures is focused on locally-occurring base pairs. However, crosslinking and proximity ligation experiments demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved complementary regions (PCCRs) in human protein-coding genes. PCCRs tend to occur within introns, suppress intervening exons, and obstruct cryptic and inactive splice sites. Double-stranded structure of PCCRs is supported by decreased icSHAPE nucleotide accessibility, high abundance of RNA editing sites, and frequent occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNAPII slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. The enrichment of 3'-ends within PCCRs raises the intriguing hypothesis that coupling between RNA folding and splicing could mediate co-transcriptional suppression of premature pre-mRNA cleavage and polyadenylation.

Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

PURPOSE: High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. PROCEDURES: The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. RESULTS: For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. CONCLUSION: The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.