Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition

Abstract

Mammalian mitochondria contain their own genome that is almost fully transcribed from both strands, generating polycistronic RNA units that are processed and matured. The mitochondrial mRNA is modified by oligo- or polyadenylation at the 3' termini, but the exact function of this post-transcriptional addition is unclear. Current debate focuses on the role of polyadenylation in transcript stability. An equally likely function that has received little attention is that, as in the cytosol of eukaryotes, polyadenylation facilitates translation in the mitochondrion. To address this issue, we have targeted cytosolic proteins to the mitochondrion, a poly(A) specific 3' exoribonuclease, mtPARN, and a poly(A)binding protein, mtPABP1. Removal of the 3' adenylyl extensions had a variable effect on mt-mRNA steady-state levels, increasing (MTND1, 2, 5) or decreasing (MTCO1, 2, RNA14) certain species with minimal effect on others (RNA7, MTND3). Translation was markedly affected, but interpretation of this was complicated by the concomitant 3' truncation of the open reading frame in most cases. Coating of the poly(A) tail by mtPABP1, however, did not lead to transcript decay but caused a marked inhibition of mitochondrial translation. These data are consistent with endogenous RNA-binding factor(s) interacting with the poly(A) to optimize mitochondrial protein synthesis.

Figure 1.

(A) Induction of mtPARN causes a variable effect on the steady-state level of mitochondrial transcripts. Stable HEK293T transfectants expressing mtLUC (lanes 1–-6), mtPARN (lanes 13–18) or the C-terminal truncated mtPARN (mtPARNΔC, lanes 7–12) were induced the number of days indicated and isolated RNA (10 µg) was subjected to northern blot analysis with the indicated probes as detailed in ‘Materials and Methods’ section. Probe from transcript encoding β-actin (ACTB) was used a loading control. (B) Northern blots were performed with the indicated probes on at least three independent RNA isolations following induction of mtPARNΔC. Quantification of steady-state mt-RNA was calculated by PhosphorImager analysis as a percentage of uninduced control. Probes were targeted against mt-rRNA or mt-mRNA encoding components of respiratory chain complexes as indicated, below. (C) Induction of mtPARN affects mitochondrial protein synthesis. Transfectants were induced for 2 days prior to in vivo mitochondrial protein synthesis with ³⁵S methionine/cysteine for 30 min as detailed. Equal amounts of mitochondrial lysate (50 µg) were separated through a 15% denaturing SDS-PAGE, the gel dried and exposed to PhosphorImager prior to visualization. Individual polypeptides were designated by their mobility (20).

Figure 2.

Expression of mtPABP1 causes a severe mitochondrial OXPHOS defect. (A) HEK293T transfectants expressing mtPABP1 or mtLUC were induced over 5 days in standard growth media and plated into 10 wells at ∼1 × 10⁵ (glucose, upper panel) or 2 × 10⁵ (galactose, bottom panel) cells/well. Ten counts were performed on duplicate wells at the indicated time points and are shown as the average ± SEM. Uninduced mtPABP1 transfectants (Glu uninduced, upper panel; Gal uninduced, lower panel) were seeded and counted in a similar fashion. (B) Samples were prepared from transfectants expressing mtLUC (L, lanes 3 and 5) or mtPABP1 (P, lanes 2 and 4) for the indicated times and subjected to western blotting following SDS- (top three panels) or blue native (bottom two panels) gel electrophoresis as detailed in ‘Materials and Methods’ section. A sample from the uninduced mtPABP1 (U_P, lane 1) is also shown. Blots of the denatured proteins were probed with antibodies to complex IV (COX2) and complex I (NDUFB8). The native gel is probed with antibodies to complexes I (NDUFA9) and III (Core 2). Loading controls are performed with antibodies to β-actin and complex II (SDH 70 kDa).

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 (U_P, 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 (U_{P 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.

Figure 4.

Occlusion of the poly(A) tail inhibits mitochondrial translation. (A) In vivo mitochondrial translation assays were performed for 15 min (lanes 1 and 2) and 45 min (lanes 3 and 4) exposure to ³⁵S methionine/cysteine after expression of mtPABP1 (lanes 2 and 4) or mtLUC (lanes 1 and 3) for 5 days as described in ‘Materials and Methods’ section. Cell lysates (50 µg) were separated through a 15% denaturing PAG, visualized and quantified using ImageQuant software following exposure to a PhosphorImager cassette. Individual polypeptides were designated on the basis of their mobility (20). To confirm equal loading, a small section of the gel is shown after exposure following Coomassie staining. (B) mtPABP1 and variants are expressed and imported into mitochondria with equal efficiency. Transfectants were induced for 3 days, mitochondria isolated and proteinase K treated as described in ‘Materials and Methods’ section. Resulting mitochondrial lysates (10 µg) were separated by 12% denaturing SDS-PAGE. Western blots were performed with anti-FLAG antibody to detect mtPABP1 and variants. Equal loading was confirmed using two antibodies specific to mitochondrial proteins, the outer membrane protein VDAC (porin) and the soluble matrix marker translation initiation factor 3 (IF3mt). Molecular weight size markers are indicated. (C) Similar in vivo labelling experiments were performed (10-min pulse) on the indicated transfectants after 3 days induction. Lysate (25 µg) was separated and visualized as described in (A). Lane 3, variant mtPAPB1(Y56V/F142V); lane 4, variant mtPABP1(F337V). (D) Induction of mtPAPB1 and variant mtPABP1(F337V) do not increase the turnover of mt-mRNA. RNA was isolated from the indicated transfectants after 0-, 3-, 5- or 8-day induction and aliquots (10 µg) subjected to northern blot analysis as detailed in ‘Materials and Methods’ section. The indicated mt-mRNA probes were used. A probe to the 12 S mt-rRNA (MTRNR1) was used as loading control.