Figure 6.
Validation of RNA Binding by Mitochondrial Enzymes
(A) Schematic of mitochondrial TCA cycle, FAO, and OXPHOS. Color scheme is as in Figure 4F. Number of known subunit variants for each OXPHOS complex is shown in brackets. NDUFA4 is considered as complex IV subunit (Balsa et al., 2012). Box to the right indicates FAO-associated enzymes.
(B) Small-scale mRNA interactome capture from HL-1 cells and western blots with antibodies against positive and negative controls (left; as in Figure 1C) as well as six endogenous TCA cycle-related enzymes (right; n = 1).
(C) Immunoprecipitation of eGFP-tagged proteins from UV-treated and control HeLa cells and 32P 5′ end labeling of crosslinked RNA fragments. Samples were processed for storage phosphor imaging (top) and western blot with anti-GFP antibody (bottom). PUM2-eGFP (at lower dose) is shown as a positive control, while eGFP alone, with or without a mitochondrial localization signal, and mock purifications from non-crosslinked cells served as negative controls (n = 1–3). Immunoprecipitation was done from WCL (left) or from purified mitochondria (right). Controls for intracellular localization of fusion proteins and mitochondrial isolation are shown in Figure S5.
See also Figure S5.