Selectivity and regulation of mRNA demethylation by iron-dependent dioxygenases - TITLE: Selectivity and regulation of mRNA demethylation by iron-dependent dioxygenases
ABSTRACT: The long-term goals of this research program are to (1) define the structural and
molecular mechanisms that control the selectivity and function of RNA demethylase enzymes, (2)
develop new chemical tools to monitor RNA demethylation in cells, and (3) understand how the
key cofactor ascorbic acid interacts with RNA demethylases and other iron-dependent
dioxygenase family members to regulate their activity. Methyl modifications on mRNA tune
transcript function, are essential for mammalian cell fate decisions, and play important roles in
the progression of many human cancers. The iron-dependent dioxygenase enzymes FTO and
AlkBH5 act as ‘erasers’ of highly abundant N6-methyladenosine (m6A) modifications found in the
mRNA body and, in the case of FTO, N6,2’-O-dimethyladenosine (m6Am) modifications found on
the 5’ mRNA cap. These RNA demethylases are overexpressed in cancers including glioblastoma
and acute myeloid leukemia, where increased demethylation activity and reduced methyl
modification levels promote tumorigenesis and cancer progression. Despite these clear links to
human disease, we currently have a poor understanding of how FTO and AlkBH5 recognize their
biological substrates, which mRNA transcripts are targeted for demethylation, and how
demethylation influences mRNA function. Furthermore, FTO and AlkBH5 belong to the non-heme
iron(II) and -ketoglutarate-dependent family of dioxygenases that require ascorbic acid (vitamin
C) as a cofactor for efficient activity, but we have almost no structure-level insights into how
ascorbic acid interacts with this diverse family of enzymes and how this physical interaction may
potentiate dioxygenase activity in cells. This proposal combines approaches from biochemistry,
structural biology, chemical biology, bioinorganic chemistry, and cell biology to determine the
structural basis for RNA demethylase selectivity, develop novel probes to map demethylation
targets across the transcriptome, and quantify and visualize the dioxygenase-ascorbic acid
interaction to understand how this cofactor regulates enzymatic activity. The results from these
proposed studies will significantly enhance our understanding of how cellular mRNA
demethylation is regulated in cells and pave the way for therapeutics that target demethylation
pathways in challenging cancers such as glioblastoma.