(A) Overview of the APEX-RIP workflow. Live cells expressing APEX2 (grey ‘pacmen’) targeted to the compartment of interest (here, the mitochondrial matrix) are incubated with the APEX substrate biotin-phenol (BP; red B: biotin). A one-minute pulse of H2O2 initiates biotinylation of proximal endogenous proteins (Rhee et al., 2013), which are subsequently crosslinked to nearby RNAs by 0.1% formaldehyde. Following cell lysis, biotinylated species are enriched by streptavidin pulldown, and coeluting RNAs are analyzed by qRT-PCR or RNA-Seq. IMM: inner mitochondrial membrane. (B) Imaging APEX2 biotinylation in situ. HEK 293T cells expressing V5-tagged mito-APEX2 were biotinylated using the APEX-RIP workflow, fixed, and stained as indicated. The bottom row is a negative control in which H2O2 treatment was omitted. Scale bars, 10 µm. TOM20 is a mitochondrial outer membrane protein; neutravidin staining detects biotinylation. (C) In situ biotinylation of the mitochondrial matrix proteome requires mito-APEX2, BP, and H2O2. Streptavidin blot analysis of whole cell lysates prepared following the protocol described in (A), or after omitting components of the APEX reaction. Arrowheads denote endogenous biotinylated proteins (Chapman-Smith and Cronan, 1999). Anti-V5 blot (bottom) detects expression of mito-APEX2. (D–E) mito-APEX-RIP efficiently recovers the mitochondrial transcriptome. (D) Gene-level RNA-Seq analysis of mito-APEX-RIP; data are the average values of three experimental replicates. Fold change is defined as (FPKMpost-enrichment/FPKMpre-enrichment); dashed lines indicate significance thresholds for fold enrichment (determined by ROC analysis, see Materials and methods) and p-values calculated by CuffDiff2 (Trapnell et al., 2013). Mitochondrial genomes encode 13 mRNAs, two rRNAs and 22 tRNAs (red). Note that three mitochondrial tRNA genes, MT-TH, MT-TL2, and MT-TG, were also enriched. See Supplementary file 1A. (E) Nucleotide-level RNA-Seq analysis of mito-APEX-RIP, mapped to the human mitochondrial genome (innermost circle). Outermost circle: reads from the full APEX-RIP protocol; middle circle: reads from the negative control. Note the enrichment of several mitochondrially-encoded tRNAs and the D-loop leader transcript. Ribosomal RNAs were removed during library preparation (see Materials and methods). See also: Figure 1—figure supplements 1,2.