Human Mitochondrial Ribosomal Protein MRPL12 Interacts Directly with Mitochondrial RNA Polymerase to Modulate Mitochondrial Gene Expression

Zhibo Wang,Justin Cotney,Gerald S Shadel

doi:10.1074/jbc.m700461200

Zhibo Wang, Justin Cotney + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m700461200

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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.

Highlights

We set out to identify proteins that interact with POLRMT that we hypothesized would be involved in new aspects of mitochondrial gene expression in humans
Identification of a Protein in HeLa Cell Mitochondrial Extracts That Binds to Human Mitochondrial RNA Polymerase (POLRMT) as Mitochondrial Ribosomal Protein L7/L12 (MRPL12)—We chose an affinity capture strategy to attempt to identify proteins that interact with POLRMT, the human mitochondrial RNA polymerase
We previously demonstrated in budding yeast that mitochondrial transcription and translation are coupled at the inner mitochondrial membrane via interactions mediated by the translation-coupling factors Nam1p and Sls1p [14]

Summary

Introduction

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 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 conserved stem loop [5, 6], representing a potential link between the transcription machinery and ribosome biogenesis and/or translation activity. The circular 16.5-kb human mtDNA molecule encodes thirteen essential protein components of the mitochondrial oxidative phosphorylation system responsible for the production of cellular ATP [15] These mRNAs are translated into protein by a dedicated set of ribosomes in the mitochondrial matrix made up of the 12 S and 16 S rRNAs, which are encoded by. We describe our finding that a conserved mitochondrial ribosomal protein is bifunctional, acting both as a component of ribosomes and of transcription-related complexes via an interaction with POLRMT

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