Mitochondrial targeting is dispensable for MDH2's RNA binding. (A) Schematic of MDH2 with the sequences relevant for mitochondrial import highlighted. MitoFates software detected the presence of amphipathic α-helices at the N terminus of both wt-MDH2 (highlighted in green as canonical MTS) and MDH2 Δ2–24 (highlighted in yellow as a cryptic MTS). At the N terminus of MDH2 Δ2–24, a Tom20-recognition motif was also detected. (B) Schematic of the wt-MDH2 and MDH2 Δ2–53 constructs. (C) Confocal immunofluorescence images showing localization of the wt-MDH2 and MDH2 Δ2–53 constructs. Images are maximum intensity projections of Z-stack images. (D) RNA-binding activity of wt-MDH2 and MDH2 Δ2–53 assessed by 2C experiments. Nucleolin is used as a positive control for RNA binding, and α-tubulin is used as a negative control. (E) Relative quantification of the RNA-binding activity of wt-MDH2 and MDH2 Δ2–53 normalized by the expression of the two constructs. Data are reported as mean ± SD, number of experiments n = 4, individual P-values are indicated according to an unpaired two-tailed t-test.
