We previously demonstrated that mammalian cells contain at least two distinct RNase P activities, one nuclear and one mitochondrial (5, 7). These enzymes were shown to have different substrate specificities and distinct molecular and enzymatic properties (5–7). Puranam and Attardi recently reported the purification of “nuclear” RNase P from mitochondrial preparations (2). However, in the discussion of their findings they made incorrect and misleading reference to previous work on mitochondrial RNase P, necessitating a reevaluation of their conclusions.
The presence of low levels of nuclear RNase P or other nuclear RNA processing factors in purified mitochondrial preparations is not a new observation, and digitonin treatment is well-known to drastically reduce the levels of these contaminants (1, 3, 7). Mitochondrial RNase P activity, however, is definitely independent of the presence or absence of nuclear RNase P or its RNA component (H1 RNA) (7). Reduction of contaminating nuclear RNase P by digitonin pretreatment of mitochondria or its complete removal from mitoplast extracts by either immunoprecipitation or purification did not result in any change of mitochondrial RNase P activity in these preparations (7).
Yet, inevitably, Puranam and Attardi (2) failed in the identification of this RNase P activity independent of H1 RNA: the use of Escherichia coli pre-tRNATyrsu3+ as a substrate throughout their purification procedure made the assay of mitochondrial RNase P activity naturally impossible. Mitochondrial RNase P as identified in 1995, which faithfully cleaves mitochondrial tRNA precursors (3–7), does not process E. coli pre-tRNATyrsu3+ (7). On the other hand, nuclear RNase P, which is capable of cleaving pre-tRNATyrsu3+, does not cleave mitochondrial pre-tRNATyr (7). Given the considerable structural differences between cytoplasmic and mitochondrial tRNAs, this particularly useful distinction of mitochondrial and nuclear RNase P by their substrate specificity is not surprising but apparently is the result of the coevolution of enzymes and substrates. Substrate recognition of both enzymes nevertheless overlaps as long as the precursors fulfill the respective structural requirements (7).
Thus, is it possible that mammalian mitochondria contain two forms of RNase P, one distinct from and one identical to the nuclear enzyme? Such a scenario of two RNase P enzymes within one cellular compartment would be without precedent and therefore requires careful evaluation. Of course, the relative and absolute levels of both enzymes in mitochondria are critical in this argument. Even in small amounts of crude mitochondrial extracts, detection of mitochondrial RNase P activity appears to be straightforward, thereby indicating a high level of enzyme (7). In contrast, nuclear RNase P activity could not be detected in mitochondrial extracts (7) but required enrichment by biochemical purification to allow assay of its activity (2). Puranam and Attardi nevertheless provided an estimate of the quantities of nuclear RNase P (H1) RNA as well as other snRNAs present in purified mitochondria (2). Taking their numbers for granted, (i) the actual levels of U snRNAs (nuclear splicing factors) and H1 RNA are in an astonishingly similar range and (ii) the estimated number of H1 RNA molecules in the mitochondrial matrix (digitonin/micrococcal nuclease resistant and “corrected for losses of mitochondrial markers”) of cells in the G2 phase of the cell cycle is only one-third of the number of mitochondria (2). The latter implies that the mitochondria of cells during other phases of the cell cycle do not contain any nuclear RNase P, which would only be possible by cell cycle stage-dependent import of nuclear RNase P followed by rapid intramitochondrial degradation or expulsion. Moreover, assuming that this scenario is correct, the numbers given by Puranam and Attardi would actually be overestimates of RNase P amounts, as their preparations were “enriched in heavy mitochondria” (2). Thus we feel that although it is impossible to formally exclude the possibility that mitochondria contain trace amounts of nuclear RNA processing factors, contamination of mitochondrial preparations during subcellular fractionation still appears to be the more plausible interpretation.
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