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

Puranam, Ram S. and Attardi, Giuseppe (2001) The RNase P Associated with HeLa Cell Mitochondria Contains an Essential RNA Component Identical in Sequence to That of the Nuclear RNase P. Molecular and Cellular Biology, 21 (2). pp. 548-561. ISSN 0270-7306. doi:10.1128/MCB.21.2.548-561.2001.

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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.

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Additional Information:Copyright © 2001, American Society for Microbiology. Received 17 July 2000/Returned for modification 16 August 2000/Accepted 19 October 2000 This work was supported by NIH grant GM11726 to G.A. We thank S. Altman for providing the NuH1 cDNA clone and S. Altman, C. Takada, A. Chomyn, K. Puranam, and P. Sethna for critically reading the manuscript. The excellent technical help of A. Drew, B. Keeley, and R. Kinzel is gratefully acknowledged. R.S.P. especially thanks W. Kibbe for the suggestion to use FPLC for enzyme purification and for his constant support and friendship. R.S.P. also owes thanks to members of G. Attardi's and J.-P. Revel's laboratory for their constant support and help.
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Deposited On:03 Apr 2006
Last Modified:08 Nov 2021 19:47

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