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. 2010 Feb 9;38(11):3732–3742. doi: 10.1093/nar/gkq068

Figure 3.

Figure 3.

mtPABP1 binds mitochondrial mRNA. (A) RNA was isolated from cells expressing mtPABP1 over an 8-day period prior to the analysis of poly(A) tail extensions as described (7). 32P-end-labelled products corresponding to the 3′ termini of MTCO2 (lanes 1–6), MTCO1 (lanes 7–10) and MTND4 (lanes 11–14) were separated through an 8% denaturing PAG and visualized by PhosphorImager and ImageQuant analysis. Zero extension is taken as the position of migration predicted on 3′ processing from the polycistronic transcript prior to any addition. Products were generated in the absence of mtPABP1 expression (UP, lanes 3, 8 and 12) or after 1 (lane 4), 5 (lanes 5, 9 and 13) or 8 (lanes 6, 10 and 14) days induction. MPAT assays were also performed on RNA isolated from cells expressing mtLUC for 8 days (L, lanes 1, 7 and 11). Lane 2 demonstrates MPAT on RNA isolated from uninduced mtPABP1 transfectants grown in 1 µg/ml ethanol for 8 days (UP ETOH). (B) Immunoprecipitated mtPABP1 associates with RNA. Immunoprecipitation of FLAG tagged mtLUC and mtPABP1 was performed as described in ‘Materials and Methods’ section. Equal volumes of RNA extracted from immunoprecipitated samples were analysed by northern blot with the indicated probes as detailed in ‘Materials and Methods’ section.