5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.

5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.

Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.

The regulatory role of N(6)-methyladenosine (m(6)A) and its nuclear binding protein YTHDC1 in pre-mRNA splicing remains an enigma. Here we show that YTHDC1 promotes exon inclusion in targeted mRNAs through recruiting pre-mRNA splicing factor SRSF3 (SRp20) while blocking SRSF10 (SRp38) mRNA binding. Transcriptome assay with PAR-CLIP-seq analysis revealed that YTHDC1-regulated exon-inclusion patterns were similar to those of SRSF3 but opposite of SRSF10. In vitro pull-down assay illustrated a competitive binding of SRSF3 and SRSF10 to YTHDC1. Moreover, YTHDC1 facilitates SRSF3 but represses SRSF10 in their nuclear speckle localization, RNA-binding affinity, and associated splicing events, dysregulation of which, as the result of YTHDC1 depletion, can be restored by reconstitution with wild-type, but not m(6)A-binding-defective, YTHDC1. Our findings provide the direct evidence that m(6)A reader YTHDC1 regulates mRNA splicing through recruiting and modulating pre-mRNA splicing factors for their access to the binding regions of targeted mRNAs.

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.

ALKBH4-dependent demethylation of actin regulates actomyosin dynamics.

Regulation of actomyosin dynamics by post-transcriptional modifications in cytoplasmic actin is still poorly understood. Here we demonstrate that dioxygenase ALKBH4-mediated demethylation of a monomethylated site in actin (K84me1) regulates actin-myosin interaction and actomyosin-dependent processes such as cytokinesis and cell migration. ALKBH4-deficient cells display elevated K84me1 levels. Non-muscle myosin II only interacts with unmethylated actin and its proper recruitment to and interaction with actin depend on ALKBH4. ALKBH4 co-localizes with the actomyosin-based contractile ring and midbody via association with methylated actin. ALKBH4-mediated regulation of actomyosin dynamics is completely dependent on its catalytic activity. Disorganization of cleavage furrow components and multinucleation associated with ALKBH4 deficiency can all be restored by reconstitution with wild-type but not catalytically inactive ALKBH4. Similar to actin and myosin knock-out mice, homozygous Alkbh4 mutant mice display early embryonic lethality. These findings imply that ALKBH4-dependent actin demethylation regulates actomyosin function by promoting actin-non-muscle myosin II interaction.

ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.

Energetic-assisted scanning thermal lithography for patterning silver nanoparticles in polymer films.

Energetic-assisted scanning thermal lithography (SThL) was demonstrated with the addition of benzoyl peroxide (BPO) for patterning silver nanoparticles. SThL samples were prepared by spin-coating poly(methyl methacrylate) (PMMA) thin films preloaded with BPO and silver nitrate precursors. Localized thermal analysis via probe heating demonstrated that the BPO decomposition in the polymer film took place at the temperature of 80 °C. Above this temperature, the thermal probe initiated the decomposition of the peroxide, which resulted in the in situ discharge of exothermal energy to compensate the joule shortage and the rapid cooling in the SThL thin film samples. The additional joule energy thermally enhanced the synthesis of silver nanoparticles, which were patterned and embedded in the PMMA thin film. Surface plasmon resonance scattering of these silver nanoparticles was observed by dark-field optical microscopy, whereas the nanoparticle distribution was examined by transmission electron microscopy. Variations in the scanning probe temperatures and peroxide concentrations were carefully investigated to optimize the thermal lithography efficiency upon the addition of energetics.

Enhancement of light scattering and photoluminescence in electrospun polymer nanofibers.

Poly(methyl methacrylate) nanofibers with desired fiber diameters that ranged from 336 to 896 nm were electrospun as light scattering and propagation materials. The light scattering behavior of these samples as a function of the fiber diameter and fiber deposition thickness was examined by UV-vis spectrophotometry, which revealed the scattering bands in the absorption spectra. The scattering bands of these nanofibers were linearly proportional to the fiber diameter, which shows good agreement with a scattering model based on the Mie theory. The light scattering and prolonged light path lengths in the nanofiber scaffolds were monitored and quantified by the photoluminescence of a fluorescent dye, Coumarin 6, which was preloaded into the polymer nanofibers. The photoluminescence after proper normalization showed a second-order dependence on the dye loading per unit area, which is significantly different from the spin-coated thin-film samples following a first-order relationship. Nonlinear photoluminescence enhancements indicated prolonged light path lengths and multiple light absorptions within the fiber scaffolds as a result of light scattering. Even with relatively broad scattering band widths, the light scattering and photoluminescence of the electrospun nanofibers exhibited considerable wavelength selectivity, especially as the scattering bands overlapped with the excitation wavelengths of the fluorescence reagent.

Accumulation of butyltin compounds in cobia Rachycentron canadum raised in offshore aquaculture sites.

Butyltin residues (monobutyltin, MBT; dibutyltin, DBT; tributyltin, TBT; tetrabutyltin, TeBT) in the sea water and in the cobia (Rachycentron canadum) from aquaculture sites located offshore of Penhu island, Taiwan, were collected and quantified. The average concentrations of MBT, DBT, TBT and TeBT in sea water were n.d.-28+/-3, 4.0+/-0.6-88+/-13, n.d.-43+/-4, and n.d.-7+/-1 ng l(-1), respectively. The total butyltin (sum of MBT, DBT, TBT, TeBT) residues in the skin, dorsal muscle, ventral muscle, dark muscle, and liver of the cobia were in the range of 72+/-12-2270+/-85, 79+/-11-688+/-33, 82+/-14-1715+/-104, 93+/-13-803+/-47, and n.d.-52,745+/-252 ng g(-1) (wet weight), respectively. Although in this study in most cases, the highest concentration of total butyltin residues was found in liver or skin, in some cases, the highest concentration was found in muscle tissue. The crude lipid content in the skin, dorsal muscle, ventral muscle, dark muscle, and liver of these cobia was in the range of 7.9+/-0.1-28+/-1%, 11.7+/-0.8-29+/-1%, 11.5+/-0.3-44+/-3%, 24.2+/-0.4-48.4+/-0.4%, and 55.7+/-0.1-87.7+/-0.4% (wet weight), respectively. The concentrations of crude lipid content, and the concentrations of total butyltin residues in these tissues were not correlated.