Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (mC) formation in mammalian RNAs is still poorly understood. However, the recent discovery of as the second gene responsible for mC presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in mC formation in mammals as well. Here, we report the discovery and characterization of three distinct mC-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute mC in specific tRNAs and that METTL8 only contributes mC to mRNA. MS analysis revealed that there is an ∼30-40% and ∼10-15% reduction, respectively, in and null-mutant cells, of mC in total tRNA, and primer extension analysis located METTL2-modified mC at position 32 of tRNA isoacceptors and tRNA We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors. , however, modified only mRNA, as determined by biochemical and genetic analyses in null-mutant mice and two human mutant cell lines. Our findings provide the first evidence of the existence of mC modification in mRNA, and the discovery of METTL8 as an mRNA mC writer enzyme opens the door to future studies of other mC epitranscriptomic reader and eraser functions.

SummaryTransforming growth factor b-activated kinase 1 (TAK1) is a key regulator of the innate immunity and the proinflammatory signaling pathway. In response to interleukin-1, tumor necrosis factor-a, and toll-like receptor agonists, it mediates the activation of the nuclear factor jB (NF-jB), c-Jun N-terminal kinase (JNK), and p38 pathways. In addition, TAK1 plays a central role in adaptive immunity, in which it mediates signaling from T-and B-cell receptors. This review will focus on recent developments and also examine the regulation of TAK1 in response to a diverse range of other stimuli including DNA damage, transforming growth factor-b, Wnt, osmotic stress, and hypoxia.2012 IUBMB IUBMB Life, 64(10): [825][826][827][828][829][830][831][832][833][834] 2012

Mammalian Pellino isoforms are phosphorylated by IRAK (interleukin receptor associated kinase) 1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. In the present paper we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDMs (bone-marrow-derived macrophages) are stimulated with LPS (lipopolysaccharide) or poly(I:C). This induction occurs via a TRIF [TIR (Toll/interleukin-1 receptor)-domain-containing adaptor-inducing interferon-β]-dependent IRAK-independent pathway and is prevented by inhibition of the IKK [IκB (inhibitor of nuclear factor κB) kinase]-related protein kinases, TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1} and IKKε. Pellino 1 is not induced in IRF3 (interferon regulatory factor 3)-/- BMDMs, and its induction is only reduced slightly in type 1 interferon receptor-/- BMDMs, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKε as bait, and show that IKKε/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288 and Ser293. Moreover, we show that the E3 ligase activity of endogenous Pellino 1 is activated in LPS- or poly(I:C)-stimulated macrophages. This occurs more rapidly than the increase in Pellino 1 mRNA and protein expression, is prevented by the inhibition of IKKε/TBK1 and is reversed by phosphatase treatment. Thus IKKε/TBK1 mediate the activation of Pellino 1's E3 ligase activity, as well as inducing the transcription of its gene and protein expression in response to TLR3 and TLR4 agonists.

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