Hiroyuki Yanagisawa

FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis

The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5′- and 3′-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.

Sets of RNA Repeated Tags and Hybridization-Sensitive Fluorescent Probes for Distinct Images of RNA in a Living Cell

Background Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. Methodology/Principal Findings Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3′-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag–probe pairs. Conclusions/Significance A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes

Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO-FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO-FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO-FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO-FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution.

5-Hydroxymethylcytosine-selective oxidation with peroxotungstate

Tungsten oxidation worked as a simple chemical reaction for the effective detection of 5-hydroxymethylcytosine in DNA, distinguishing it from its epigenetic precursors, 5-methylcytosine and unmethylated cytosine. The tungsten-oxidation product obtained from 5-hydroxymethylcytosine was trihydroxylated thymine and was detected as a cleavage band in gel electrophoresis after treatment with hot piperidine.

Hybridization-sensitive fluorescence control in the near-infrared wavelength range

A series of near-infrared fluorescent probes were designed based on the concept of emission control caused by interdye excitonic interaction. The fluorescent probes showed very weak emission in the unhybridized state, whereas they emitted near-infrared fluorescence after hybridization with the complementary nucleic acid. The hybridization-dependent switching of fluorescence emission made it possible to monitor mRNA in human cells in the range of near-infrared wavelengths.

Design and Synthesis of Caged Fluorescent Nucleotides and Application to Live-cell RNA Imaging

A binary photocontrolled nucleic acid probe that contains a nucleotide modified with one photolabile nitrobenzyl unit and two hybridization‐sensitive thiazole orange units has been designed for area‐specific fluorescence imaging of RNA in a cell. The synthesized probe emitted very weak fluorescence regardless of the presence of the complementary RNA, whereas it showed hybridization‐sensitive fluorescence emission at 532 nm after photoirradiation at 360 or 405 nm for uncaging. Fluorescence suppression of the caged probe was attributed to a decrease in the duplex‐formation ability. Caged fluorescent nucleotides with other emission wavelengths (622 and 724 nm) were also synthesized in this study; they were uncaged by 360 nm irradiation, and emitted fluorescence in the presence of the complementary RNA. Such probes were applied to area‐specific RNA imaging in a cell. Only probes in the defined irradiation area were activated by uncaging irradiation, and subnuclear mRNA diffusion in a living cell was monitored.

Cy5-Conjugated Hybridization-Sensitive Fluorescent Oligonucleotides for Ratiometric Analysis of Nuclear Poly(A)+ RNA

Subnuclear poly(A)(+) RNA localization in living mammalian cells was visualized by ratiometric analysis using hybridization-sensitive fluorescent oligonucleotide probes. Probes were oligonucleotides, which contained a Cy5 fluorescent dye at the strand end and a thiazole orange double-labeled nucleotide inside strand. A ratiometric analysis using poly(A)-targeting probes revealed a distribution of the probe itself as red fluorescence and localization of the target RNA sequence in cell nuclei as green fluorescence. The fluorescence of the subnuclear poly(A)(+) RNA hybridized with the poly(A)-targeting probes was observed as puncta in interchromatin areas.

Ligand‐Incorporation Site in 5‐Methylcytosine‐Detection Probe Modulating the Site of Osmium Complexation with the Target DNA

ICON Probes, short DNA strands containing an adenine linked to a bipyridine ligand, formed an interstrand cross‐link with 5‐methylcytosine located opposite the modified adenine in the presence of an osmium oxidant. The location of a bipyridine‐tethered adenine in the probes varied the selectivity of the reactive base. An ICON probe where the modified adenine was located at the probe center showed a 5‐methylcytosine‐selective osmium complexation, whereas an ICON probe with the modified adenine at the strand end exhibited high reactivity towards thymine as well as 5‐methylcytosine. The modulation of reactive bases by the incorporation of a bipyridine‐tethered adenine site made facilitates design of ICON probes for the fluorometric detection of 5‐methylcytosine.

Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit

Fluorescent probes for the detection of a double-stranded DNA were prepared by labeling a triplex-forming DNA oligonucleotide with a thiazole orange (TO) dimer unit. They belong to ECHO (exciton-controlled hybridization-sensitive fluorescent oligonucleotide) probes which we have previously reported. The excitonic interaction between the two TO molecules was expected to effectively suppress the background fluorescence of the probes. The applicability of the ECHO probes for the detection of double-stranded DNA was confirmed by examining the thermal stability and photophysical and kinetic properties of the DNA triplexes formed by the ECHO probes.

Synthesis of exciton-controlled fluorescent probes for RNA imaging

The excitonic interaction of thiazole orange dyes is known to suppress fluorescence emission. We have developed hybridization-sensitive fluorescent probes utilizing the excitonic interaction of two thiazole orange dyes connected to the probes. Here, we report nuclease-resistant hybridization-sensitive probes for long-term intracellular RNA imaging.