Role of FAST Kinase Domains 3 (FASTKD3) in Post-transcriptional Regulation of Mitochondrial Gene Expression

Abstract

The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria.

Keywords: FASTKD3; RNA; RNA metabolism; gene expression; mitochondria; post-transcriptional regulation; translation; translation control.

FIGURE 1.

Generation of FASTKD3^−/− cells. A, schematic illustration of FASTKD3 gene targeting with pAAV-MCS-FASTKD3 plasmid. N, NotI; S, SalI; E, EcoRI. Exons are indicated by black boxes. LoxP sites are depicted with triangles, with the orientation indicated by the direction of the triangle. BSD, blasticidin-S-deaminase resistance marker. B, representative PCR analysis for site-specific integration. Positions of the primers used for screening are designated by arrows in A, and expected size differences for PCR products are indicated. C, Western blotting analysis of whole-cell extracts from wild type and FASTKD3^−/− with the indicated antibodies.

FIGURE 2.

Mitochondrial RNA analysis in FASTKD3^−/− cells. A, RNA isolated from the indicated cell lines was analyzed by Northern blotting hybridization with probes specific for the mitochondrial mRNAs and rRNAs, and as a loading control, with probes for 7SL (signal recognition particle RNA). A representative experiment done in triplicates is shown. B, intensities of radioactive bands on Northern blots shown in A were quantified by densitometric analysis using ImageJ software. Data were normalized to 7SL RNA levels and presented relative to the wild type control (+/+, set as 1). Values represent the means ± S.E. (n = 3). *, p < 0.05, **, p < 0.01, ***, p < 0.001. C, analysis of mtRNA half-lives and mtDNA content in FASTKD3^−/− cells. Total RNA was isolated at different times up to 6 h after treatment with ethidium bromide. Steady-state levels of mitochondrial mRNAs were quantified by qRT-PCR as described under “Experimental Procedures.” The half-life of each mitochondrial mRNA was calculated using the formula t_½ = ln2/λ, where λ is the slope of mRNA decay. Data are expressed as means ± S.D. (n = 5).

FIGURE 3.

Analysis of the domains required for the decay acceleration activity of FASTKD3. A, RAP domain protein sequence homology analysis among the different FASTK members was performed using ClustalW program. White letters on a black background highlight identical amino acids. White letters on a gray background highlight different but conserved amino acids. The red box indicates the five partially conserved amino acids at the C terminus end of the RAP domain of FASTK family members. Amino acid positions of the RAP domain boundaries are indicated. NCBI reference sequence identifiers for the aligned sequences are: FASTK (NP_006703), FASTKD1 (NP_001308975), FASTKD2 (NP_001129665), FASTKD3 (NP_076996), FASTKD4 (NP_004740), and FASTKD5 (NP_068598). B, RNA isolated from wild type, FASTKD3^−/− cells, and FASTKD3^−/− cells reconstituted with full-length (FL) FASTKD3-FLAG-HA, FASTKD3ΔRAP-FLAG-HA, or FASTKD3Δ646–650-FLAG-HA (by lentiviral vector-mediated transduction) was analyzed by Northern blotting with the indicated probes. The lower two panels show Western blotting analysis of whole-cell lysates using antibodies against HA or β-actin (loading control). C, confocal microscopy analysis of U2OS cells transiently overexpressing full-length FASTKD3-GFP, FASTKD3ΔRAP-GFP, or FASTKD3Δ646–650-GFP. Mitochondria were stained with MitoTracker (CMX-ROS), and Hoechst 33258 was used for nuclei staining.

FIGURE 4.

Mitochondrial protein synthesis in FASTKD3^−/− cells. A, de novo mitochondrial protein synthesis by metabolic labeling in FASTKD3^−/− cells (left panel). Two independent FASTKD3^−/− cell lines (clone #1 and clone #2) were incubated in the presence of 200 μCi of [³⁵S]cysteine and [³⁵S]methionine for 1 h after the addition of emetine (100 μg/ml). Proteins were separated by SDS-PAGE in a 12–20% linear gradient gel. The gel was stained with Coomassie brilliant blue (CBB), and radioactivity was detected using a phosphorimaging device. Complete lanes from the same phosphorimaging device exposure were rearranged so that each clone and its corresponding wild type control were juxtaposed. This is indicated by leaving a space between the lanes. In the right panel, band intensities in FASTKD3^−/− cells were determined by densitometric analysis, and data are expressed relative to those of wild type cells (set as 1). Values represent the means ± S.D. (n = 3). **, p < 0.01. B, representative Western blot showing expression of COX1 and other MRC subunits on whole cell lysates from wild type and FASTKD3^−/− cells (left panel). The right panel shows quantification of Western blotting signals in FASTKD3^−/− cells. Values were normalized to β-actin (loading control) and presented relative to wild type (set as 1). Values represent the means ± S.E. (n = 7).**, p < 0.01. C, representative Western blot of four independent experiments showing expression of COX1 in cell lysates from wild type, FASTKD3^−/−, and FASTKD3^−/− cells reconstituted with full-length (FL) FASTKD3-FLAG-HA, FASTKD3ΔRAP-FLAG-HA, or FASTKD3Δ646–650-FLAG-HA (by lentiviral vector-mediated transduction).

FIGURE 5.

Assembly and activity of MRC complexes in FASTKD3^−/− cells. A, BN-PAGE and Western blotting analyses of digitonin-solubilized mitochondrial extracts from wild type and FASTKD3^−/− cells. Membranes were incubated with the indicated oxidative phosphorylation subunits. I+III+IV, SC containing CI, CIII, and CIV; I+III, SC containing CI and CIII; and III+IV, SC containing CIII and CIV. Data are representative of two independent experiments out of four. B, the specific activities of complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), and complex IV (cytochrome c oxidase) were measured in cell lysates, and then normalized to citrate synthase activity and expressed as: (nmol/min/mg of protein)/(specific activity of citrate synthase) × 100. Data are presented as means ± S.E. The number of independent experiments and the p value using Mann Whitney U test are indicated. ns, not significant.