Biology subjects

Gamage, S. T.

Publications and source records attributed to Gamage, S. T..

3 recordsLinked to original sources

bioRxiv2023

Rewiring of RNA methylation by the oncometabolite fumarate in renal cell carcinoma

Metabolic reprogramming is a hallmark of cancer that facilitates changes in many adaptive biological processes. Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) lead to fumarate accumulation and cause hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC is a rare, inherited disease characterized by the development of non-cancerous smooth muscle tumors of the uterus and skin, and an increased risk of a highly metastatic and aggressive form of kidney cancer. Fumarate has been shown to inhibit 2-oxyglutarate-dependent dioxygenases (2OGDDs) involved in the hydroxylation of HIF1, as well as in DNA and histone demethylation. However, the link between fumarate accumulation and changes in RNA post-transcriptional modifications has not been defined. Here, we determine the consequences of fumarate accumulation on the activity of different members of the 2OGDD family targeting RNA modifications. By evaluating multiple RNA modifications in patient-derived HLRCC cell lines, we show that mutation of FH selectively alters the activity of demethylases acting upon N6-methyladenosine (m6A), while the demethylase acting upon N1-methyladenosine (m1A) and 5-formylcytosine (f5C) in mitochondrial RNA are unaffected. The observation that metabolites modulate specific subsets of RNA-modifying enzymes offers new insights into the intersection between metabolism and the epitranscriptome.

molecular biology↗

bioRxiv2022

Conditional covalent lethality driven by oncometabolite accumulation

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a cancer predisposition syndrome driven by mutation of the tumor suppressor fumarate hydratase (FH). Inactivation of FH causes accumulation of the electrophilic oncometabolite fumarate. In the absence of methods for reactivation, tumor suppressors can be targeted via identification of synthetic lethal interactions using genetic screens. Inspired by recent advances in chemoproteomic target identification, here we test the hypothesis that the electrophilicity of the HLRCC metabolome may produce unique susceptibilities to covalent small molecules, a phenomenon we term conditional covalent lethality. Screening a panel of chemically diverse electrophiles we identified a covalent ligand, MP-1, that exhibits FH-dependent cytotoxicity. Synthesis and structure-activity profiling identified key molecular determinants underlying the molecules effects. Chemoproteomic profiling of cysteine reactivity together with clickable probes validated the ability of MP-1 to engage an array of functional cysteines, including one lying in the Zn-finger domain of the tRNA methyltransferase enzyme TRMT1. TRMT1 overexpression rescues tRNA methylation from inhibition by MP-1 and partially attenuates the covalent ligands cytotoxicity. Our studies highlight the potential for covalent metabolites and small molecules to synergistically produce novel synthetic lethal interactions and raise the possibility of applying phenotypic screening with chemoproteomic target identification to identify new functional oncometabolite targets.

biochemistry↗

bioRxiv2021

Protonation-Dependent Sequencing of 5-Formylcytidine in RNA

Chemical modification of cytidine in non-coding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remains unknown due to a lack of sensitive site-specific sequencing technologies. Here we report a protonation-dependent sequencing reaction for detection of 5-formylcytidine (5fC) and 5-carboxycytidine (5caC) in RNA. First, we evaluate how protonation combined with electron-withdrawing substituents alters the molecular orbital energies and reduction of modified cytidine nucleosides, highlighting 5fC and 5caC as reactive species. Next, we apply this reaction to detect these modifications in synthetic oligonucleotides as well as endogenous human tRNA. Finally, we demonstrate the utility of our method to characterize a patient-derived model of 5fC-deficiency, where it enables facile monitoring of both pathogenic loss and exogenous rescue of NSUN3-dependent 5fC within the wobble base of human mitochondrial tRNAMet. These studies showcase the ability of protonation to enhance the reactivity and sensitive detection of 5fC in RNA, and provide a molecular foundation for applying optimized sequencing reactions to better understand the role of oxidized RNA cytidine nucleobases in disease.

biochemistry↗