Drosophila melanogaster LRPPRC2 is involved in coordination of mitochondrial translation

Abstract

Members of the pentatricopeptide repeat domain (PPR) protein family bind RNA and are important for post-transcriptional control of organelle gene expression in unicellular eukaryotes, metazoans and plants. They also have a role in human pathology, as mutations in the leucine-rich PPR-containing (LRPPRC) gene cause severe neurodegeneration. We have previously shown that the mammalian LRPPRC protein and its Drosophila melanogaster homolog DmLRPPRC1 (also known as bicoid stability factor) are necessary for mitochondrial translation by controlling stability and polyadenylation of mRNAs. We here report characterization of DmLRPPRC2, a second fruit fly homolog of LRPPRC, and show that it has a predominant mitochondrial localization and interacts with a stem-loop interacting RNA binding protein (DmSLIRP2). Ubiquitous downregulation of DmLrpprc2 expression causes respiratory chain dysfunction, developmental delay and shortened lifespan. Unexpectedly, decreased DmLRPPRC2 expression does not globally affect steady-state levels or polyadenylation of mitochondrial transcripts. However, some mitochondrial transcripts abnormally associate with the mitochondrial ribosomes and some products are dramatically overproduced and other ones decreased, which, in turn, results in severe deficiency of respiratory chain complexes. The function of DmLRPPRC2 thus seems to be to ensure that mitochondrial transcripts are presented to the mitochondrial ribosomes in an orderly fashion to avoid poorly coordinated translation.

Figure 1.

Subcellular localization of DmLRPPRC2. (A) Fluorescence confocal microscopy images showing HeLa cells (n = 15) expressing a DmLRPPRC2-GFP fusion protein and counterstained with the mitochondrial marker MitoTracker Deep Red. Scale bar: 10 μm. (B) Subcellular fractionation showing the presence of DmLRPPRC2 in the mitochondrial fraction. Subcellular fractions were separated by SDS-PAGE followed by western blot analysis to detect histone H3 (a nuclear marker), tubulin (a cytosolic marker), VDAC (a mitochondrial marker) and DmLRPPRC2. See also Supplementary Figure S1A and B.

Figure 2.

Efficient ubiquitous downregulation of DmLrpprc2 expression. (A) qRT-PCR analyses of DmLrpprc2 and DmLrpprc1 mRNA levels in the Lrpprc2RNAi#1 line measured in controls (white and light gray bars) and DmLrpprc2 KD (dark gray bars) third-instar larvae and 6-day-old flies (n = 4–5). Error bars indicate mean ± 1 SD. Student's t-test was applied, *P < 0.05, ^**P < 0.01, ^***P < 0.001. (B) Western blot experiments to detect DmLRPPRC2 and DmLRPPRC1 in the Lrpprc2RNAi#1 line. Mitochondrial protein extracts (40 μg) from control and DmLrpprc2 KD third-instar larvae were separated by standard SDS-PAGE, followed by western blot analysis with antibodies against DmLRPPRC2, DmLRPPRC1 and VDAC, the latter used as reference for loading. (C) qRT-PCR analyses of DmLrpprc2 and DmLrpprc1 mRNA levels in the Lrpprc2RNAi#2 line measured in controls (white and light gray bars) and DmLrpprc2 KD (black bars) third-instar larvae and 6-day-old flies (n = 4–5). Error bars indicate mean ± 1 SD. Student's t-test was applied, *P < 0.05, ^**P < 0.01, ^***P < 0.001. (D) Western blot experiments to detect DmLRPPRC2 and DmLRPPRC1 in the Lrpprc2RNAi#2 line. Mitochondrial protein extracts (40 μg) from control and DmLrpprc2 KD third-instar larvae were separated by standard SDS-PAGE, followed by western blot analysis with antibodies against DmLRPPRC2, DmLRPPRC1 and VDAC, the latter used as reference for loading. See also Supplementary Figure S2.

Figure 3.

Larval size, eclosion, climbing index and lifespan analyses. (A) Body size comparison at the 6-day-old third-instar larval stage showing reduced size for DmLrpprc2 KD larvae compared to controls. Scale bar: 1 mm. (B) Eclosion rates of the Lrpprc2RNAi#1 line (left) and the Lrpprc2RNAi#2 line (right) in control (white and light gray bars) and DmLrpprc2 KD (dark gray and black bars) individuals. Data are shown relative to the w;;daGAL4/+ control line. Error bars indicate mean ± 1 SD. Student's t-test was applied, *P < 0.05, ^**P < 0.01, ^***P < 0.001. (C) Climbing test on males of the Lrpprc2RNAi#2 line, showing impaired climbing skills of 13-day-old DmLrpprc2 KD individuals (n = 12) in comparison to controls (n = 5–6). Error bars indicate mean ± 1 SD. Mann–Whitney U test was applied, *P < 0.05. (D) Survival curves of the Lrpprc2RNAi#1 line (upper graph) and the Lrpprc2RNAi#2 line (lower graph), showing shortened lifespan for DmLrpprc2 KD individuals in comparison to controls. Error bars indicate mean ± 1 SD. Long-rank test was applied.

Figure 4.

Steady-state levels and activities of the OXPHOS complexes. (A) Western blot analysis of the Lrpprc2RNAi#1 line (left side) and the Lrpprc2RNAi#2 line (right side) performed on 20–40 μg whole-body protein extracts from controls and DmLrpprc2 KD third-instar larvae and 6-day-old adults. Protein extracts were separated by standard SDS-PAGE followed by western blot analysis with antibodies against the nuclear-encoded subunit NDUFS3 of complex I, the α-subunit of complex V and VDAC, the latter used as reference for loading. (B) BN-PAGE combined with complex I, complex IV and complex V in-gel activity analyses on the Lrpprc2RNAi#1 line and the Lrpprc2RNAi#2 line. BN-PAGE was performed on 75 μg for complex I, 100 μg for complex IV and 150 μg for complex V of mitochondrial protein extracts from control and DmLrpprc2 KD third-instar larvae. The assembly status of complex V (right panel) is black because of color inversion. Loading controls are provided by Coomassie staining to assess the total mitochondrial protein content per sample (only for complex V, left panel) and western blot analysis of VDAC protein levels in mitochondrial protein lysates collected prior to BN-PAGE gel loading. The position of complex I (CI), complex IV (CIV), complex V dimers (CV₂), complex V monomers (CV₁) and complex V subassembled components (subCV_a and subCV_b) are indicated by arrows.

Figure 5.

Biochemical measurement of respiratory chain function. (A) Respiratory chain enzyme activities, normalized to the CS activity, were used to assess the activities of complex I (NADH coenzyme Q reductase, reported as I), complex II (succinate dehydrogenase, reported as II), complexes I–III (NADH coenzyme Q reductase-cytochrome c reductase, reported as I–III), complexes II–III (succinate dehydrogenase-cytochrome c reductase, reported as II–III) and complex IV (cytochrome c oxidase, reported as IV) in isolated mitochondria from control (white and gray bars) and DmLrpprc2 KD (black bars) third-instar larvae of the Lrpprc2RNAi#2 line (n = 6–12). Error bars indicate mean ± SEM. Student's t-test was applied, *P < 0.05, ^**P < 0.01, ^***P < 0.001. (B) Oxygen consumption measurements normalized to protein content in permeabilized control (white and gray bars) and DmLrpprc2 KD (black bars) third-instar larvae and thoraces from 6-day-old adults of the Lrpprc2RNAi#2 line (n = 5). Respiratory rate analyses were performed using substrates delivering electrons at the level of complex I (CPI), complex II and glycerol-3-phosphate dehydrogenase (SUCC-G3P), or by using combined substrates delivering electrons at the level of complex I, II and glycerol-3-phosphate dehydrogenase (CPI-SUCC-G3P). Error bars indicate mean ± SEM. Student's t-test was applied, *P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 6.

Steady-state levels of mitochondrial mRNAs, rRNAs and tRNAs. (A) qRT-PCR analyses on mitochondrial mRNAs normalized to the nuclear ribosomal protein 49 (rp49) transcript levels in control (white and gray bars) and DmLrpprc2 KD (black bars) third-instar larvae or 6-day-old adults of the Lrpprc2RNAi#2 line (n = 4–5). Error bars indicate mean ± 1 SD. Student's t-test was applied, *P < 0.05, ^**P < 0.01, ^***P < 0.001. (B) Northern blot analyses of steady-state levels of mitochondrial mRNAs, rRNAs and tRNAs, normalized to the rp49 transcript levels, in third-instar larvae of the Lrpprc2RNAi#2 line. (C) Quantification of northern blots shown in panel B. Error bars indicate mean ± 1 SD. Student's t-test was applied, *P < 0.05, ^**P < 0.01, ^***P< 0.001. (D) Northern blot analyses of steady-state levels of mitochondrial mRNAs, rRNAs and tRNAs, normalized to the rp49 transcript levels, in 6-day-old adults of the Lrpprc2RNAi#2 line. (E) Quantification of northern blots shown in panel D. Error bars indicate mean ± 1 SD. Student's t-test was applied, *P < 0.05, ^**P < 0.01, ^***P < 0.001. See also Supplementary Figures S3 and S4.

Figure 7.

Analysis of mtDNA levels, de novo transcription and in organello translation. (A) Mitochondrial de novo transcription performed in isolated mitochondria from third-instar larvae of the Lrpprc2RNAi#2 line. De novo synthesized mitochondrial transcripts were labeled with α-³²P-dUTP and the cytB transcript detected by northern blot analyses was used as loading control, given that its steady-state levels were not different in DmLrpprc2 KD and control individuals. Quantification of de novo transcription was performed by normalizing the profile of each lane to the cytb mRNA steady-state levels. The values reported for each genotype correspond to the average of two replicates. (B) qPCR analyses of steady-state levels of mtDNA in control (white and gray bars) and DmLrpprc2 KD (black bars) third-instar larvae or 6-day-old adults of the Lrpprc2RNAi#2 line (n = 4–5). Error bars indicate mean ± 1 SD. Student's t-test was applied, *P < 0.05, ^**P < 0.01, ^***P < 0.001. (C) Mitochondrial in organello translation performed on mitochondrial protein extracts from third-instar larvae of the Lrpprc2RNAi#2 line. Samples were collected 1 h after ³⁵S-methionine-labeling (pulse, right panel, left side) or 3 and 5 h after cold methionine addition (chase, right panel, right side) and separated in standard SDS-PAGE gels. Loading controls are provided by Coomassie staining to assess the total mitochondrial protein content per sample (left panel) and western blot analysis of VDAC protein levels in mitochondrial protein extracts collected after pulse and chase experiments. Asterisks mark the polypeptides whose levels are increased in DmLRPPRC2 KD samples compared to controls.

Figure 8.

The interaction between mRNAs and the ribosome, as determined by sucrose density gradients. Mitochondrial RNAs were loaded on the top of a sucrose density gradient to assess the co-migration with the ribosome. Mitochondrial lysates from control (left side) and DmLrpprc2 KD (right side) third-instar larvae of the Lrpprc2RNAi#2 line were analyzed. Mitochondrial transcripts were quantified through qRT-PCR analyses by using TaqMan or SYBR probes. The relative RNA abundance in each fraction is represented as the percentage relative to the total RNA abundance in the 20 fractions. The small (28S) ribosomal subunit, the large (39S) ribosomal subunit and the assembled (55S) ribosome are indicated by arrows. The 12S rRNA sedimentation profile was used as marker for the migration of the 28S and the 55S ribosome particles. The 16S rRNA sedimentation profile was used as a marker for the migration of the 39S and 55S ribosome particles. See also Supplementary Figure S5.