Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control.

N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia

Slow Growth and Increased Spontaneous Mutation Frequency in Respiratory Deficient afo1- Yeast Suppressed by a Dominant Mutation in ATP3

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the repiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency

Handbuch der gnadenreichen und verdienstvollen Bruderschaft des hochheiligsten Herzens Jesu und des unbefleckten Herzens Mariä : errichtet und eingeführt in der Pfarrkirche zum heiligen Andreas in Hofendorf im Jahre des Herrn 1848 : ein allgemein brauchbares, schönes Gebetbuch

mit allerhöchster Genehmigung, bischöflicher Erlaubniß und päpstlicher Bestätigung von Wolfgang Geltinger, Pfarrer in Hosendor

Activation of c-myc promoter P1 by immunoglobulin κ gene enhancers in Burkitt lymphoma: functional characterization of the intron enhancer motifs kB, E box 1 and E box 2 and of the 3' enhancer motif PU.

eregulated expression of the proto-oncogene c-myc in Burkitt lymphoma (BL) cells carrying a t(2;8) translocation is mediated by a synergistic interaction of the translocated immunoglobulin (Ig) kappa gene intron (kappa Ei) and 3' (kappa E3') enhancers and characterized by a strong activation of the promoter P1, We have investigated the functional role of distinct kappa enhancer sequence motifs in P1 activation on both minichromosomes and reporter gene constructs, Stable and transient transfections of BL cells revealed critical roles of the kappa Ei and kappa E3' elements kappa B and PU, respectively, Joint mutation of kappa B and PU completely abolished P1 activity, implying that an interaction of kappa B- and PU-binding factors is essential for the enhancer synergism, Mutation of the E box 1 and E box 2 motifs markedly decreased P1 activity in transient but not in stable transfection experiments. Co-expression of the NF-kappa B subunit p65(RelA) and Sp1, an essential factor for P1 transcription, in Drosophila melanogaster SL2 cells synergistically enhanced promoter activity. Our results support a model which proposes cross-talk between promoter and enhancer binding factors as the basic mechanism for kappa enhancer-mediated c-myc activation in BL cell

Probing the Recognition of Post-Translational Modifications by Combining Sortase-Mediated Ligation and Phage-Assisted Selection

Reversible post-translational modifications (PTMs) are key regulators of protein function and modulate a multitude of protein–protein interactions in signal transduction networks. Here, we describe a strategy for determining the modification preferences of PTM-binding proteins with only minimal protein amounts that can be obtained by immunoprecipitation from mammalian cell lysates. This method bases on the combination of sortase-mediated ligation and phage-assisted selection strategies. This method can be used to analyze the type of modification that mediates the interaction as well as the influence of the amino acids flanking the modification sites. We have demonstrated the applicability of this method by probing the interaction of phosphorylated tyrosine and serine residues with their respective binding domains