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Molecular and Cellular Biology, January 2001, p. 548-561, Vol. 21, No. 2
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.2.548-561.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The RNase P Associated with HeLa Cell Mitochondria Contains an Essential RNA Component Identical in Sequence to That of the Nuclear RNase P

Ram S. Puranamdagger and Giuseppe Attardi*

Division of Biology, California Institute of Technology, Pasadena, California 91125

Received 17 July 2000/Returned for modification 16 August 2000/Accepted 19 October 2000

The mitochondrion-associated RNase P activity (mtRNase P) was extensively purified from HeLa cells and shown to reside in particles with a sedimentation constant (~17S) very similar to that of the nuclear enzyme (nuRNase P). Furthermore, mtRNase P, like nuRNase P, was found to process a mitochondrial tRNASer(UCN) precursor [ptRNASer(UCN)] at the correct site. Treatment with micrococcal nuclease of highly purified mtRNase P confirmed earlier observations indicating the presence of an essential RNA component. Furthermore, electrophoretic analysis of 3'-end-labeled nucleic acids extracted from the peak of glycerol gradient-fractionated mtRNase P revealed the presence of a 340-nucleotide RNA component, and the full-length cDNA of this RNA was found to be identical in sequence to the H1 RNA of nuRNase P. The proportions of the cellular H1 RNA recovered in the mitochondrial fractions from HeLa cells purified by different treatments were quantified by Northern blots, corrected on the basis of the yield in the same fractions of four mitochondrial nucleic acid markers, and shown to be 2 orders of magnitude higher than the proportions of contaminating nuclear U2 and U3 RNAs. In particular, these experiments revealed that a small fraction of the cell H1 RNA (of the order of 0.1 to 0.5%), calculated to correspond to ~33 to ~175 intact molecules per cell, is intrinsically associated with mitochondria and can be removed only by treatments which destroy the integrity of the organelles. In the same experiments, the use of a probe specific for the RNA component of RNase MRP showed the presence in mitochondria of 6 to 15 molecules of this RNA per cell. The available evidence indicates that the levels of mtRNase P detected in HeLa cells should be fully adequate to satisfy the mitochondrial tRNA synthesis requirements of these cells.

* Corresponding author. Mailing address: Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125. Phone: (626) 395-4930. Fax: (626) 449-0756. E-mail: attardig{at}seqaxp.bio.caltech.edu.

dagger Present address: Department of Medicine (Neurology), Duke University Medical Center, Durham, NC 27710.

Molecular and Cellular Biology, January 2001, p. 548-561, Vol. 21, No. 2
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.2.548-561.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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