Figure 2.

The mitochondrial targeting peptide is necessary but not sufficient to guide asymmetric localization of mRNA. mRNA localization was assessed statistically by FISH analyses (more than 100 cells), as indicated in supplementary Fig S1 online. (A) The absence of MTS-coding sequence reduces ATP2 mRNA asymmetric localization. (B) MTS-coding sequences alone are not able to guide heterologous mRNAs to the vicinity of mitochondria. The full-length COX4-coding sequence was also unable to promote asymmetric mRNA localization, in agreement with previous studies (Saint-Georges et al, 2008). (C) Heterologous MTS fully restore the asymmetric localization of an MTS-deleted version of ATP2 mRNA. MTS from cytoplasmically localized ATP16 and COX4 was also able to confer asymmetric localization to the ATP2 ORF sequence. (D) Single mRNA-resolution FISH analysis using the four hybrid mRNAs described above. The numbers 3, 9, 10 and 11 correspond to the constructs in (B) and (C). Cells were hybridized by using probes against LacZ (labelled with Cy3) and 16S rRNA (labelled with Cy5). Scale bars, 1 μm. (E) ATP2, ATP3 and ATP4 mRNA asymmetric localization is MTS-dependent. DAPI, diamidino-2-phenylindole; FISH, fluorescent in situ hybridization; mRNA, messenger RNA; mt rRNA, mitochondrial ribosomal RNA; MTS, mitochondrial targeting peptide.