Figure 1.

Mitochondria-specific knockout of dRNaseZ. (A) N-terminal sequences of RNase Z^L from different species. The mitochondria import probabilities (right) were calculated using MitoProt. Two initiating methionines are in red. The putative MTS is in bold. (B) The N-termini and C-termini of proteins encoded by RNZ^WT and RNZ^ΔMTS constructs. Methionines are in red and the mutated amino acid is in blue. V5-tags are attached to the C-termini of WT and mutant proteins. (C,D) Immunostaining of S2 cultured cells (C) and testes (D) expressing RNZ^WT and RNZ^ΔMTS proteins. In merge: mitochondria (Mitotracker) are red, DNA (Hoechst) is blue, and anti-V5 is green. (C) S2 cells transfected with plasmids encoding RNZ^WT or RNZ^ΔMTS proteins. Arrowhead indicates a non-transfected cell. (D) Onion stage spermatids from transgenic males carrying the genRNZ^WT or genRNZ^ΔMTS construct. Note that in (C,D) RNZ^WT co-localizes with mitochondria (yellow), while RNZ^ΔMTS does not. (E) Northern blot analysis of RNA samples from WT control (lane 1 and 7), RNZ^ED24 KO (lane 2 and 8) and RNZ^ΔMTS (lane 3–5 and 9–11), or RNZ^WT (lane 6 and 12) rescued KO larvae. The internal-probe detects mature nuclear Histidine (nHis) and mitochondrial Tyrosine (mtTyr) tRNAs; the 3′-probe detects primary transcripts and processing intermediates.