[show abstract][hide abstract] ABSTRACT: The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.Cell Research advance online publication 10 January 2014; doi:10.1038/cr.2014.3.
[show abstract][hide abstract] ABSTRACT: DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5mC to 5hmC in vivo, and increase 5hmC in 5751 genes in cells. 5hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5hmC increase, and a delivery of labile iron increased the 5hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.
Nucleic Acids Research 11/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ∼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells. We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, diRNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. Knock down of Dicer or Ago2 in human cells reduces DSB repair. Our findings reveal a conserved function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules directing chromatin modifications or the recruitment of protein complexes to DSB sites to facilitate repair.
[show abstract][hide abstract] ABSTRACT: The maintenance of genomic stability requires accurate genome replication, repair of DNA damage, and the precise segregation of chromosomes in mitosis. GEN1 possesses Holliday junction resolvase activity in vitro and presumably functions in homology driven repair of DNA double strand breaks. However, little is currently known about the cellular functions of human GEN1. In the present study we demonstrate that GEN1 is a novel centrosome associated protein and we characterize the various phenotypes associated with GEN1 deficiency. We identify an N-terminal centrosome localization signal in GEN1, which is required and sufficient for centrosome localization. We report that GEN1 depletion results in aberrant centrosome numbers associated with the formation of multiple spindle poles in mitosis, an increased number of cells with multi-nuclei, increased apoptosis and an elevated level of spontaneous DNA damage. We find homologous recombination severely impaired in GEN1 deficient cells, suggesting that GEN1 functions as a Holliday junction resolvase in vivo as well as in vitro. Complementation of GEN1 depleted cells with various GEN1 constructs revealed that centrosome association but not catalytic activity of GEN1 is required for preventing centrosome hyper-amplification, formation of multiple mitotic spindles, and multi-nucleation. Our findings provide novel insight into the biological functions of GEN1 by uncovering an important role of GEN1 in the regulation of centrosome integrity.
PLoS ONE 01/2012; 7(11):e49687. · 3.73 Impact Factor