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.The core machinery responsible for specific initiation of transcription from human mtDNA promoters has been elucidated. The minimal requirement is a three-component complex comprising a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNAbinding protein h-mtTFA/TFAM, and two transcriptional coactivator proteins, h-mtTFB1 and h-mtTFB2 (1). The mtTFB factors interact directly with a carboxyl-terminal tail of h-mt-TFA and bridge interactions between the promoter-bound h-mtTFA and POLRMT to facilitate specific initiation (2). Despite these advances, our understanding of the nature of mitochondrial transcription complexes in vivo remains limited, especially in mammalian systems. For example, whether other factors interact with POLRMT to regulate transcription initiation and/or elongation or to couple additional RNA-related activities to transcription have not been determined. However, interesting in this regard, h-mtTFB1 and h-mtTFB2 are members of a conserved family of rRNA methyltransferases (3, 4) and can methylate small subunit rRNAs at a conserve...

Mitochondrial Ribosomal Protein L12 selectively associates with human mitochondrial RNA polymerase to activate transcription

“…We showed previously that MRPL12 interacts with POLRMT in vitro and increases mitochondrial transcript levels when overexpressed in HeLa cells (34). To investigate further the role of MRPL12 in mitochondrial gene expression in vivo, we knocked it down using lentiviral-induced shRNA in HeLa and HEK293T cells.…”

“…In addition to their rRNA subunits, the large and small ribosomal subunits are each composed of multiple MRPs. We identified previously that MRPL7/L12 (referred to as MRPL12 from this point forward) binds directly to POLRMT and modulates mitochondrial gene expression when overexpressed in HeLa cells (34), providing a functional link between mitochondrial ribosomes/ translation and transcription in human mitochondria. Here we have investigated this link further and conclude, based on both in vivo and in vitro evidence, that it is a "free" pool of MRPL12 (i.e., not associated with assembled ribosomes) that interacts with POLRMT to directly activate mitochondrial transcription.…”

Core human mitochondrial transcription apparatus is a regulated two-component system in vitro

“…In Figs 1-3, the linear DNA templates for simultaneously analyzing transcription from HSP1 and LSP (Fig. 1A) were PCR products corresponding to human mtDNA sequence 242-825 (LSP3) or 317-825 (LSP3.1) cloned into the pGEMT-EZ (Promega, Inc.) (26). For use in the transcription reactions, the mtDNA template was liberated from the vector by digestion with EcoR1 (LSP3) or NotI (LSP3.1), gelpurified, and quantified by OD 260 .…”

“…However, the nature of transcription complexes in vivo remains largely undetermined and the involvement of other factors is clear. For example, the MTERF family of proteins regulates various aspects of transcription (4,(23)(24)(25) and human MRPL12, in addition to its role in mitochondrial ribosomes, binds directly to POLRMTand activates transcription in mitochondrial lysates (26).…”

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

“…The L12 stalk is required for translation, because it recruits to the ribosome the auxiliary translation factors, in particular translational GTPases, such as initiation factor 2, elongation factors Tu and G, and release factor 3 or their eukaryotic homologues [2][3][4][5][6][7][8] , and accelerates GTPase activity of some of them 3,[9][10][11] . The mitochondrial homologue of L12, MRPL12, not only has an important role in translation but also regulates the transcription in mitochondria 12 . Variations in the L12 sequence between species provide the specificity for the interactions with translation factors: the replacement of the L12-L10 stalk in Escherichia coli ribosomes with its eukaryotic counterpart (P0/P1/P2 stalk) makes the ribosome to bind eukaryotic, rather than bacterial elongation factor 13 .…”