Exploring the Role of DNMT1 in RNA m5C Methylation (RNA-BS-SEQ DNMT1 knockdown)

DNA methyltransferase 1 (DNMT1) is an enzyme known for DNA methylation maintenance. However, point mutations in its RFTS domain lead to late-onset neurodegeneration such as the autosomal dominant cerebellar ataxia-deafness and narcolepsy (ADCA-DN) disorder. Here we demonstrated that wild-type DNMT1 also has the capability to bind to mRNA transcripts and facilitate 5-methylcytosine (m5C) RNA methylation by recruiting NOP2/Sun RNA methyltransferase 2 (NSUN2). RNA m5C methylation, in turn, promotes RNA stability for those genes modulating mitochondrial function. When DNMT1 RFTS domain is mutated in the case of ADCA-DN disorder, it triggers aberrant DNMT1-RNA interaction and significantly elevated m5C RNA methylation and RNA stability for a portion of metabolic genes. Consequently, increased levels of metabolic RNA transcripts contribute to cumulative oxidative stress, mitochondrial dysfunction, and neurological symptoms. Collectively, our results highlight a novel role for DNMT1 in regulating both DNA and RNA methylation as well as mitochondrial function, shedding light on the pathogenic mechanism of DNMT1 mutation-induced neurodegeneration. Overall design: HeLa cells were transfected with small interfering RNA (siRNA) targeting DNMT1 using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's instructions. For the a-Amanitin treatment, HeLa cells in culture were treated with 15 µg/ml of the RNA polymerase II and III inhibitor a-Amanitin (Santa Cruz) for 12 h. Subsequently, the cells were washed twice with PBS, collected, and processed for RNA-bisulfite sequencing (RNA-BS-seq).