An Upstream Open Reading Frame and the Context of the Two AUG Codons Affect the Abundance of Mitochondrial and Nuclear RNase H1

Abstract

RNase H1 in mammalian cells is present in nuclei and mitochondria. Its absence in mitochondria results in embryonic lethality due to the failure to amplify mitochondrial DNA (mtDNA). Dual localization to mitochondria and nuclei results from differential translation initiation at two in-frame AUGs (M1 and M27) of a single mRNA. Here we show that expression levels of the two isoforms depend on the efficiency of translation initiation at each AUG codon and on the presence of a short upstream open reading frame (uORF) resulting in the mitochondrial isoform being about 10% as abundant as the nuclear form. Translation initiation at the M1 AUG is restricted by the uORF, while expression of the nuclear isoform requires reinitiation of ribosomes at the M27 AUG after termination of uORF translation or new initiation by ribosomes skipping the uORF and the M1 AUG. Such translational organization of RNase H1 allows tight control of expression of RNase H1 in mitochondria, where its excess or absence can lead to cell death, without affecting the expression of the nuclear RNase H1.

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We thank Tom Dever (NICHD) for helpful comments and suggestions on the manuscript. Nicole Follmer initiated some of these experiments. Xiongfong (Jack) Chen provided help in database searches. We thank P. Sarnow and M. S. Carter, Stanford University, for luciferase plasmids.

This work was supported in part by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health. Yutaka Suzuki was a Japan Society for the Promotion of Science Research Fellow in Biomedical and Behavioral Research at the National Institutes of Health. J. Bradley Holmes is a National Institutes of Health-Cambridge University Biomedical Research Scholar. Michal Minczuk and Ian Holt are supported by the U.K. Medical Research Council.