Human mitochondrial ribosomal protein MRPL12 interacts directly with mitochondrial RNA polymerase to modulate mitochondrial gene expression

Abstract

The core human mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNA-binding protein h-mtTFA/TFAM, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2 that also have rRNA methyltransferase activity. Recapitulation of specific initiation of transcription in vitro can be achieved by a complex of POL-RMT, h-mtTFA, and either h-mtTFB1 or h-mtTFB2. However, the nature of mitochondrial transcription complexes in vivo and the potential involvement of additional proteins in the transcription process in human mitochondria have not been extensively investigated. In Saccharomyces cerevisiae, transcription and translation are physically coupled via the formation of a multiprotein complex nucleated by the binding of Nam1p to the amino-terminal domain of mtRNA polymerase (Rpo41p). This model system paradigm led us to search for proteins that interact with POLRMT to regulate mitochondrial gene expression in humans. Using an affinity capture strategy to identify POL-RMT-binding proteins, we identified mitochondrial ribosomal protein L7/L12 (MRPL12) as a protein in HeLa mitochondrial extracts that interacts specifically with POLRMT in vitro. Purified recombinant MRPL12 binds to POLRMT and stimulates mitochondrial transcription activity in vitro, demonstrating that this interaction is both direct and functional. Finally, from HeLa cells that overexpress FLAG epitope-tagged MRPL12, increased steady-state levels of mtDNA-encoded transcripts are observed and MRPL12-POLRMT complexes can be co-immunoprecipitated, providing strong evidence that this interaction enhances mitochondrial transcription or RNA stability in vivo. We speculate that the MRPL12 interaction with POLRMT is likely part of a novel regulatory mechanism that coordinates mitochondrial transcription with translation and/or ribosome biogenesis during human mitochondrial gene expression.

FIGURE 1. Affinity capture of mitochondrial ribosomal protein L12 (MRPL12) in association with POLRMT, the human mitochondrial RNA polymerase

A, purification of His-tagged POLRMT by nickel-affinity and heparin-agarose chromatography. Shown are Coomassie-stained SDS-PAGE gels of representative fractions from a typical purification of recombinant POLRMT from E. coli lysate after nickel-affinity chromatography (Ni-NTA) and subsequent heparin-agarose (heparin) chromatography. The ∼130-kDa POLRMT band is indicated. B, results of affinity capture of POLRMT-binding proteins from HeLa cell mitochondrial lysates. Shown is a silver-stained SDS-PAGE gel of proteins eluted from control DHFR beads (left lane) or POLRMT beads (right lane). The major ∼20-kDa band that co-purified with POLRMT is labeled MRPL12, based on the mass spectroscopy-based identification performed on the excised band. C, mass spectroscopy-based peptide analysis identified the 20-kDa band in panel B as MRPL12. The seven peptides unambiguously identified as MRPL12 are underlined (or overlined) on the primary sequence of the human MRPL12 polypetide (amino acids 1–198) and labeled 1–7. The gray box indicates the mitochondrial localization sequence (MLS) of MRPL12. Note that no peptides were identified in this region as would be predicted if the MLS were cleaved off upon import.

FIGURE 2. Recombinant MRPL12 binds directly to POLRMT in vitro

A, purification of recombinant MRPL12 from E. coli. Shown is a Coomassie-stained SDS-PAGE gel of three stages of the GST-based purification of MRPL12. The GST-MRPL12 fusion protein after glutathione-agarose chromatography is shown in the left lane, cleavage of the fusion protein with thrombin to remove the GST tag is shown in the middle lane, and the MRPL12 protein after separation from the cleaved GST peptide is shown in the right lane (see “Materials and Methods” for details). The latter fraction was used in all of the in vitro experiments in this report involving recombinant MRPL12. B, shown are silver-stained gels that represent the results of an in vitro binding experiment. The same amount of recombinant MRPL12 (input) was added to beads alone (left lane; negative control), DHFR beads (middle lane; negative protein control), or POLRMT beads (right lane). After several stringency washes, in the resulting “pull down” of bound proteins, MRPL12 only was found associated with POLRMT beads (indicated by the arrowhead).

FIGURE 3. Recombinant MRPL12 stimulates mitochondrial transcription from the mitochondrial LSP and HSP1 promoters in vitro

A, shown is an autoradiogram of labeled RNA transcripts from an in vitro mitochondrial transcription assay. The two run-off transcripts generated from initiation at the human mtDNA LSP and HSP promoters are labeled. The presence (+) or absence (−) of ∼200 ng of partially purified POLRMT fraction from HeLa cell mitochondria (POLRMT), recombinant h-mtTFA (40 ng), or recombinant MRPL12 (200 ng) in each reaction is indicated at the bottom. B, immunodepletion of MRPL12 from the recombinant MRPL12 preparation eliminates its transcription stimulatory activity. Shown is the LSP run-off transcript only. The presence (+) or absence (−) of a partially purified POLRMT fraction from HeLa cell mitochondria (POLRMT), recombinant MRPL12, or the antibody used (preimmune, “pre”; immune “L12”) to treat the recombinant MRPL12 preparation prior to addition to the transcription reaction is indicated at the bottom.

FIGURE 4. Co-immunoprecipitation of POLRMT-MRPL12 complexes from HeLa cells

Shown are Western blots of the input (top panels) and anti-FLAG antibody-mediated immunoprecipitations (bottom panels) of MRPL12 from HeLa whole cell lysates from an empty vector transfected negative control cell line (V) or a FLAG-tagged MRPL12 overexpression cell line (L12). The input lanes were probed with the FLAG antibody to detect FLAG-tagged MRPL12 and with antibodies that recognize HSP60 (a mitochondrial matrix marker) or COX1 (a mitochondrial inner membrane protein) as controls. The immunoprecipitations were probed with these same antibodies, as well as with a POLRMT antibody to assay for co-immunoprecipitation of POLRMT by FLAGtagged MRPL12.

FIGURE 5. Overexpression of MRPL12 in HeLa cells enhances the steadystate level of mtDNA-encoded transcripts

Shown is a Northern analysis of the mtDNA-encoded mRNAs from HeLa cells. The top is an autoradiogram of the blots probed for either the ND2 or the ND6 mRNA in three biological replicates of RNA isolated from empty vector control (vector) or MRPL12 overexpression HeLa cells (same lines as described in Fig. 4). The 28 S cytoplasmic rRNA from the ethidium-stained gel is also shown and was used as the loading control. The relative abundance of the ND2 and ND6 RNA was quantified based on the 28 S loading control and graphed (bottom). The ratio of the ND2/28 S or ND6/28 S in the vector control cell lines (V) was given a value of 1.0 (white bars) and used to normalize the signals from the MRPL12 overexpression lines (L12). Thus, the gray bars represent the fold up-regulation of ND2 and ND6 mRNA in the MRPL12 overexpression cell line. All data represent the average of six values ± S.D. (brackets), that is, two experiments done in triplicate (one representative experiment is shown).