Abstract 1444 Investigating the intersection of one carbon metabolism and mitochondrial genome maintenance

Mitochondria contain their own DNA (mtDNA) that carries the genetic instructions for proteins required for ATP synthesis. The objective of this research is to further understand the regulation of mtDNA transcription and genome maintenance through the study of proteins that interact with the mitochondrial genome. Approximately 50 mitochondrial proteins (termed nucleoid proteins) have been mapped to mtDNA. Interestingly, four of these nucleoid proteins are also central to one-carbon metabolism (MTHFD1L, SHMT2, ALDH1L2, PRDX5). One-carbon metabolism is a biosynthetic process which produces nucleotides and nitrogenous bases that serve as building blocks for DNA and RNA. We hypothesize that the proximity of one-carbon metabolic proteins to mtDNA is required to relay nutrient status signals that regulate mtDNA maintenance and expression. To investigate the impact of the one-carbon metabolism nucleoid proteins on the mitochondrial genome, the expression levels of MTHFD1L, SHMT2, ALDH1L2, and PRDX5 were altered in HeLa cells using siRNA knockdown or transient overexpression. qPCR was used to assess mtDNA content, as well as RNA transcript levels for specific mitochondrial genes. Results indicate that overexpressing ALDH1L2 and PRDX5 decrease mtDNA content. siRNA knockdown has variable effects, with loss of MTHFD1L decreasing mtDNA content, and loss of ALDH1L2 increasing mtDNA content. Altered expression levels of one-carbon proteins have varied effects on mitochondrial transcripts. To determine the nature of the interaction of one-carbon metabolism nucleoid proteins with mtDNA, confocal microscopy and protein-DNA binding assays were performed. Immunofluorescence microscopy was used to assess the colocalization of the four proteins with mtDNA. Protein binding assays were used to determine whether the proteins bind directly to mtDNA and whether binding occurs in a sequence-specific manner. Preliminary DNA binding and immunofluorescence results indicate that SHMT2 binds directly to mtDNA. This material is based upon work supported by the National Science Foundation under Grant No. MCB CAREER 2141375 and the Herbert H. and Grace A. Dow Foundation. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.